TLR2 mediates immunity to experimental cysticercosis

Abstract

Information concerning TLR-mediated antigen recognition and regulation of immune responses during helminth infections is scarce. TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of viruses, bacteria, fungi and parasites. Here, we evaluated the role of TLR2 in a Taenia crassiceps cysticercosis model. We compared the course of T. crassiceps infection in C57BL/6 TLR2 knockout mice (TLR2⁻/⁻) with that in wild type C57BL/6 (TLR2⁺/⁺) mice. In addition, we assessed serum antibody and cytokine profiles, splenic cellular responses and cytokine profiles and the recruitment of alternatively activated macrophages (AAMφs) to the site of the infection. Unlike wild type mice, TLR2⁻/⁻ mice failed to produce significant levels of inflammatory cytokines in either the serum or the spleen during the first two weeks of Taenia infection. TLR2⁻/⁻ mice developed a Th2-dominant immune response, whereas TLR2⁺/⁺ mice developed a Th1-dominant immune response after Taenia infection. The insufficient production of inflammatory cytokines at early time points and the lack of Th1-dominant adaptive immunity in TLR2⁻/⁻ mice were associated with significantly elevated parasite burdens; in contrast, TLR2⁺/⁺ mice were resistant to infection. Furthermore, increased recruitment of AAMφs expressing PD-L1, PD-L2, OX40L and mannose receptor was observed in TLR2⁻/⁻ mice. Collectively, these findings indicate that TLR2-dependent signaling pathways are involved in the recognition of T. crassiceps and in the subsequent activation of the innate immune system and production of inflammatory cytokines, which appear to be essential to limit infection during experimental cysticercosis.

Keywords: TLR2; Taenia crassiceps; alternatively activated macrophages; cysticercosis.

Figure 1

TLR2^-/- C57BL/6 mice are unable to efficiently control Taenia crassiceps infection. The course of i.p. T. crassiceps infection in TLR2^-/- and TLR2^+/+ mice following infection with 20 cysticerci. Data are expressed as means ± SE of data from 4-6 mice per group. *P < 0.05 and **P < 0.01 when comparing TLR2^-/- mice to TLR2^+/+ mice at the same time point of infection. Similar results were observed in two independent experiments.

Figure 2

Kinetics of antibody production during T. crassiceps infection in TLR2^-/- (closed squares) and TLR2^+/+ (open squares) mice. A) Anti-T. crassiceps IgG2a. B) Anti-T. crassiceps IgG1. Sera were obtained from the tail vein of each mouse at the indicated time points. ELISA plates were coated with 0.5 μg/well of T. crassiceps cysticerci soluble extract. Data are presented as means ± SE (n= 4 animals) and are representative of two independent experiments. *P < 0.05 when comparing TLR2^-/- mice to TLR2^+/+ mice at the same time point.

Figure 3

Cell proliferation and Th1-/Th2-related cytokine production in infected TLR2^-/- and wild-type mice. A) Proliferative responses were assessed by measuring ³H-thymidine incorporation in splenocytes stimulated with soluble Taenia crassiceps antigen (TcAg) and Concanavalin A (ConA) at different time points post-infection. Splenocytes from mice infected with Taenia crassiceps for 8 weeks were stimulated with TcAg or ConA in vitro. Production of (B) IL-12, (C) IL-4 and (D) IFN-γ was quantified by ELISA. Data are expressed as means ± SEM and are representative of two independent experiments with similar results. *P < 0.05 and **P < 0.01. n= 4-6 mice per group. TLR, Toll-like receptor.

Figure 4

Systemic Th2-biased responses in TLR2^-/- mice infected with Taenia crassiceps metacestodes. The kinetics of cytokine production after infection with 20 metacestodes was assessed by measuring (A) IFN-γ detection in sera. (B) TLR2^-/- mice displayed increased IL-4 levels as the infection progressed; (C) IL-10 and (D) IL-12 levels in sera were inconsistent. The data shown are representative of two independent experiments, with four mice per group. The error bars indicate standard deviations. *P < 0.05 and **P < 0.01. n= 4-6 mice per group. TLR, Toll-like receptor.

Figure 5

Taenia crassiceps infection induces peritoneal recruitment of AAMΦs after 8 weeks of infection in TLR2^-/- mice. RT-PCR analysis of adherent peritoneal macrophages recovered from animals infected with 20 metacestodes 0, 4, 6 and 8 weeks p.i. Increased expression of genes associated with AAMΦs, such as Arginase1, RELM-α, Ym1, PD-L1 and PD-L2, was detected only in TLR2^-/- T. crassiceps-infected mice. The data shown are representative of two independent experiments with four mice per group.

Figure 6

Taenia crassiceps infection induces peritoneal recruitment of AAMΦs after 8 weeks of infection only in TLR2^-/- mice. Flow cytometric analysis of peritoneal exudate cells recovered from animals infected with 20 metacestodes 8 weeks p.i. (A) Surface expression of F4/80 and MMR (CD206). (B) Surface expression of F4/80 and PDL-1, (C) F4/80 and PDL-2 and (D) F4/80 and OX40L. The dot plots represent four mice per group, and the data are representative of two independent experiments.

Figure 6

Taenia crassiceps infection induces peritoneal recruitment of AAMΦs after 8 weeks of infection only in TLR2^-/- mice. Flow cytometric analysis of peritoneal exudate cells recovered from animals infected with 20 metacestodes 8 weeks p.i. (A) Surface expression of F4/80 and MMR (CD206). (B) Surface expression of F4/80 and PDL-1, (C) F4/80 and PDL-2 and (D) F4/80 and OX40L. The dot plots represent four mice per group, and the data are representative of two independent experiments.

Figure 6

Taenia crassiceps infection induces peritoneal recruitment of AAMΦs after 8 weeks of infection only in TLR2^-/- mice. Flow cytometric analysis of peritoneal exudate cells recovered from animals infected with 20 metacestodes 8 weeks p.i. (A) Surface expression of F4/80 and MMR (CD206). (B) Surface expression of F4/80 and PDL-1, (C) F4/80 and PDL-2 and (D) F4/80 and OX40L. The dot plots represent four mice per group, and the data are representative of two independent experiments.

Figure 6

Taenia crassiceps infection induces peritoneal recruitment of AAMΦs after 8 weeks of infection only in TLR2^-/- mice. Flow cytometric analysis of peritoneal exudate cells recovered from animals infected with 20 metacestodes 8 weeks p.i. (A) Surface expression of F4/80 and MMR (CD206). (B) Surface expression of F4/80 and PDL-1, (C) F4/80 and PDL-2 and (D) F4/80 and OX40L. The dot plots represent four mice per group, and the data are representative of two independent experiments.