Toll-like receptor 4 mediates tolerance in macrophages stimulated with Toxoplasma gondii-derived heat shock protein 70

Abstract

Peritoneal macrophages (PMs) from toll-like receptor 4 (TLR4)-deficient and wild-type (WT) mice were responsive to recombinant Toxoplasma gondii-derived heat shock protein 70 (rTgHSP70) and natural TgHSP70 (nTgHSP70) in NO release, but those from TLR2-, myeloid differentiation factor 88 (MyD88)-, and interleukin-1R-associated kinase 4 (IRAK4)-deficient mice were not. Polymyxin B did not inhibit PM activation by TgHSP70 and nTgHSP70 from WT and TLR4-deficient mice, while it inhibited PM activation by lipopolysaccharide. Pretreatment of PMs from WT but not from TLR4-deficient mice with rTgHSP70 resulted in suppression of NO release on restimulation with rTgHSP70. Similarly, pretreatment of PMs from WT but not TLR4-deficient mice with nTgHSP70 resulted in suppression of NO release on restimulation with nTgHSP70. Polymyxin B did not inhibit rTgHSP70- and nTgHSP70-induced tolerance of PMs from TLR4-deficient mice. Furthermore, PMs from WT mice increased suppressor of cytokine-signaling-1 (SOCS-1) expression after restimulation with rTgHSP70, while those from TLR4-deficient mice did not. Phosphorylation of JNK and I-kappaBalpha occurred in rTgHSP70-induced tolerance of PMs from TLR4-deficient mice, but not in that from WT mice. These data indicated that TgHSP70 signaling mechanisms were mediated by TLR2, MyD88, and IRAK4, but not by TLR4. On the other hand, signaling of TgHSP70-induced tolerance was mediated by TLR4, and the expression of SOCS-1 suppressed the TLR2 signaling pathway.

FIG. 1.

TgHSP70 induces NO release. (A) PMs from WT mice were harvested. Serial dilutions of rTgHSP70, rTgHSP30, or rSAG1 were added to the culture of PMs and were present for the entire cultivation time of 24 h. (B) PMs from TLR4-, TLR2-, MyD88-, and IRAK4-deficient and WT mice were stimulated for 24 h in the presence of 2 μg/ml of rTgHSP70, rTgHSP30, or rSAG1. NO₂⁻ was measured 24 h posttreatment by Griess reaction. A P value of <0.05 was taken as significant. Three to four mice comprised each group in each experiment, which was performed in triplicate. The error bars indicate standard deviations.

FIG. 2.

TLR2 regulates TgHSP70-induced NO release. (A and B) PMs from TLR4-deficient (closed circles), TLR2-deficient (closed triangles), MyD88-deficient (closed diamonds), and IRAK4-deficient (open squares) and WT (closed squares) mice were harvested. Serial dilutions of rTgHSP70 (A) or LPS (B) were simultaneously added to the culture of PMs and were present for the entire cultivation time of 24 h. NO₂⁻ was measured 24 h posttreatment by Griess reaction. (C and D) PMs from TLR4-, TLR2-, and MyD88-deficient and WT mice were stimulated for 8 h in the presence of rTgHSP70 (2 μg/ml; C) or LPS (1 ng/ml; D), and iNOS mRNA expression was determined by RT-PCR. The data are representative of three independent experiments. (E) Ten micrograms per milliliter of polymyxin B (PB) was added to the culture with rTgHSP70 (2 μg/ml) or LPS (1 ng/ml) and was present for the entire cultivation time of 24 h posttreatment by Griess reaction. (F) Ten microliters of TgNCR C2 was added to the culture with rTgHSP70 (2 μg/ml) or LPS (1 ng/ml) and was present for the entire cultivation time of 24 h posttreatment by Griess reaction. (G) Ten micrograms per milliliter of polymyxin B (PB) was added to the culture with nTgHSP70 (2 μg/ml) and was present for the entire cultivation time of 24 h posttreatment by Griess reaction. (H) SDS-PAGE analysis of rTgHSP70 and nTgHSP70. Recombinant TgHSP70 (lane 1) and nTgHSP70 (lane 2) were run on a 10% SDS-PAGE with a molecular size marker (M; kDa). P < 0.05 was taken as significant. Three to four mice comprised each group in each experiment, which was performed in triplicate. The error bars indicate standard deviations.

FIG. 3.

TLR4 induces TgHSP70-induced NO release tolerance. PMs from TLR4-, TLR2-, and MyD88-deficient and WT mice were stimulated for 24 h with rTgHSP70 (2 μg/ml) (A), LPS (30 ng/ml) (A), or nTgHSP70 (2 μg/ml) (B). Ten micrograms per milliliter of polymyxin B was added to the culture with rTgHSP70, LPS, or nTgHSP70 and was present for the entire cultivation time of 24 h posttreatment. Then, the cells were washed twice and restimulated for an additional 24 h with the same concentration of rTgHSP70 (A), LPS (A), or nTgHSP70 (B). Polymyxin B was added to the culture with rTgHSP70, LPS, or nTgHSP70 and was present for the entire cultivation time of 24 h posttreatment by Griess reaction. A P value of <0.05 was taken as significant. Three to four mice comprised each group in each experiment, which was performed in triplicate.

FIG. 4.

TLR2 regulates TgHSP70-induced TNF-α mRNA expression and tolerance. (A) PMs from TLR4-, TLR2-, and MyD88-deficient and WT mice were stimulated for 8 h in the presence of rTgHSP70 (2 μg/ml), and TNF-α mRNA expression was determined by RT-PCR. Ten micrograms per milliliter of polymyxin B (PB) was added to the culture with rTgHSP70 and was present for the entire cultivation time of 8 h posttreatment. (B) PMs from TLR4-, TLR2-, and MyD88-deficient and WT mice were stimulated for 24 h with rTgHSP70 (2 μg/ml) and polymyxin B (10 μg/ml). Then, the cells were washed twice and restimulated for an additional 8 h with the same concentrations of rTgHSP70 and polymyxin B. A P value of <0.05 was taken as significant. Three to four mice comprised each group in each experiment, which was performed in triplicate.

FIG. 5.

Suppression of TgHSP70 response by SOCS-1 in mouse PMs. PMs from TLR4-, TLR2-, and MyD88-deficient and WT mice were stimulated with rTgHSP70 (2 μg/ml) or LPS (30 ng/ml). Ten micrograms per milliliter of polymyxin B was added to the culture with rTgHSP70 or LPS and was present for the entire cultivation time of 12 h posttreatment; then, the PMs were restimulated after being washed for an additional 8 h with the same concentration of polymyxin B-treated rTgHSP70 and LPS. Total RNA was isolated, and the SOCS-1 and IFN-β mRNA expression levels from PMs from mice were investigated by RT-PCR. The data are representative of three independent experiments. stim, stimulation.

FIG. 6.

Effects of pretreatment with rTgHSP70 on phosphorylation of I-κΒα and JNK. PMs from TLR4-, TLR2-, and MyD88-deficient and WT mice were stimulated for 12 h with rTgHSP70 (2 μg/ml) and polymyxin B (PB; 10 μg/ml). Then, the cells were washed twice and restimulated for an additional 60 min with the same concentrations of rTgHSP70 and polymyxin B. Protein lysates from the cells were obtained as described in Materials and Methods. The phosphorylation levels of Iκ-Bα and JNK were ascertained by Western blotting. Each set of data is representative of three independent experiments.

FIG. 7.

Mechanism of TgHSP70 tolerance in PMs from WT mice. TgHSP70 stimulation leads to TLR2 activation and the pathway of TLR2-MyD88-IRAK4-TRAF6-IKK-mediated phosphorylation of I-κB. These signaling cascades produce NO. Simultaneously, TgHSP70-stimulated TLR4 mediates IFN-β activation that suppresses NF-κB-inducing kinase (NIK) (18). Inhibition of these signaling cascades by IFN-β is possibly mediated by SOCS-1 (7) and limits the production of NO.