Signal transduction, gene transcription, and cytokine production triggered in macrophages by exposure to trypanosome DNA

Abstract

Activation of a type I cytokine response is important for early resistance to infection with Trypanosoma brucei rhodesiense, the extracellular protozoan parasite that causes African sleeping sickness. The work presented here demonstrates that trypanosome DNA activates macrophages to produce factors that may contribute to this response. Initial results demonstrated that T. brucei rhodesiense DNA was present in the plasma of C57BL/6 and C57BL/6-scid mice following infection. Subsequently, the effect of trypanosome DNA on macrophages was investigated; parasite DNA was found to be less stimulatory than Escherichia coli DNA but more stimulatory than murine DNA, as predicted by the CG dinucleotide content. Trypanosome DNA stimulated the induction of a signal transduction cascade associated with Toll-like receptor signaling in RAW 264.7 macrophage cells. The signaling cascade led to expression of mRNAs, including interleukin-12 (IL-12) p40, IL-6, IL-10, cyclooxygenase-2, and beta interferon. The treatment of RAW 264.7 cells and bone marrow-derived macrophages with trypanosome DNA induced the production of NO, prostaglandin E2, and the cytokines IL-6, IL-10, IL-12, and tumor necrosis factor alpha. In all cases, DNase I treatment of T. brucei rhodesisense DNA abolished the activation. These results suggest that T. brucei rhodesiense DNA serves as a ligand for innate immune cells and may play an important contributory role in early stimulation of the host immune response during trypanosomiasis.

FIG. 1.

Cell-free T. brucei rhodesiense DNA is detectable in the plasma of C57BL/6 and scid mice. Wild-type C57BL/6 (A) and C57BL/6-scid (B) mice were injected intraperitoneally with 1 × 10⁵ trypanosomes. Tail blood (12 μl) was collected over the course of infection. Parasitemia was measured using a hemacytometer. Plasma was prepared by centrifugation and filtration. DNA was isolated from the plasma, and trypanosome DNA was measured using real-time PCR specific for the trypanosome bip gene (see Materials and Methods). Data are represented as the means and standard errors of the results from three independent experiments consisting of six mice per experiment.

FIG. 2.

T. brucei rhodesiense DNA stimulates NO production in RAW 264.7 cells in a dose-dependent manner. RAW 264.7 cells were primed with 20 U/ml IFN-γ for 24 h followed by treatment with IFN-γ alone, CpG ODN, E. coli DNA, T. brucei rhodesiense DNA, and murine DNA at a range of concentrations for 24 h. Cell-free supernatant fluid was collected, and the production of NO was measured using the Griess reaction. Results are presented as the means ± standard errors of the means of triplicate cultures.

FIG. 3.

Trypanosome DNA activation of macrophages is not due to endotoxin contamination. BMMP from BALB/c and C.C3-Tlr4lps-d (TLR4^−/− congenic BALB/c) mice were primed with 20 U/ml IFN-γ for 24 h, followed by 24 h of treatment with medium alone (untreated), IFN-γ alone (IFN-γ), 100 ng/ml purified LPS (pLPS), 10 μg/ml CpG-containing ODN (CpG ODN), or 100 μg/ml trypanosome DNA (tryp DNA). Cell-free supernatant fluid was collected, and the production of NO was measured using the Griess reaction. Results are presented as the means ± standard errors of the means of triplicate cultures.

FIG. 4.

T. brucei rhodesiense DNA activates selected signal transduction components associated with TLR9 receptor signaling in macrophages. RAW 264.7 cells were stimulated with medium alone (untreated), 10 μg/ml CpG-containing ODN (CpG ODN), 10 μg/ml non-CpG-containing ODN (non-CpG ODN), 100 μg/ml trypanosome DNA (tryp DNA), and 100 μg/ml DNase I-treated trypanosome DNA (DNase) for 30 min (A) and for 20, 40, 60, and 120 min (B). Cell lysates (25 μg/lane) were subjected to Western blot analysis using antibodies specific for the active form of ERK 1/2, p38, and JNK 1/2. An antibody specific for IκB-α was used to detect degradation of cellular IκB-α. Blots were reprobed with antibodies specific for the inactive form of p38, ERK 1/2, pan-JNK, or actin.

FIG. 5.

Stimulation of RAW 264.7 cells with T. brucei rhodesiense DNA results in the induction of pro- and anti-inflammatory gene expression. RAW 264.7 cells were primed with 20 U/ml IFN-γ for 24 h, followed by treatment with medium alone, CpG ODN, non-CpG ODN, trypanosome (tryp) DNA, or DNase (as described in the legend to Fig. 4) for 6 h. Total cellular RNA was then collected as described in Materials and Methods, and mRNA expression was analyzed for selected genes using RT-PCR. Amplified products were separated by gel electrophoresis and detected by ethidium bromide staining. G3PDH was used as a control for equal cDNA loading.

FIG. 6.

Stimulation with T. brucei rhodesiense DNA results in the production of immunomodulatory molecules. RAW 264.7 cells (A) and BMMP (B) were primed with IFN-γ for 24 h. Then the cells were treated for 24 h with CpG ODN, non-CpG ODN, trypanosome (tryp) DNA, and DNase, as described in the legends to Fig. 4 and 5. Where indicated, BMMP were also cotreated with 20 μg/ml non-CpG ODNs (iODNs) and trypanosome DNA (iODN tryp) as a control. Following stimulation, cell-free supernatant fluids were collected, and cytokines and PGE₂ were measured by sandwich ELISA. NO production was measured using the Griess reaction. All results were normalized to cellular protein levels. Levels of detectable products are presented as the means ± standard errors of the means of results from triplicate cultures. Significant differences (P < 0.05) were found between the product levels induced by trypanosome DNA and all negative controls.