Toll-like receptors participate in macrophage activation and intracellular control of Leishmania (Viannia) panamensis

Abstract

Toll-like receptors (TLRs) play a central role in macrophage activation and control of parasitic infections. Their contribution to the outcome of Leishmania infection is just beginning to be deciphered. We examined the interaction of Leishmania panamensis with TLRs in the activation of host macrophages. L. panamensis infection resulted in upregulation of TLR1, TLR2, TLR3, and TLR4 expression and induced tumor necrosis factor alpha (TNF-α) secretion by human primary macrophages at comparable levels and kinetics to those of specific TLR ligands. The TLR dependence of the host cell response was substantiated by the absence of TNF-α production in MyD88/TRIF(-/-) murine bone marrow-derived macrophages and mouse macrophage cell lines in response to promastigotes and amastigotes. Systematic screening of TLR-deficient macrophages revealed that TNF-α production was completely abrogated in TLR4(-/-) macrophages, consistent with the increased intracellular parasite survival at early time points of infection. TNF-α secretion was significantly reduced in macrophages lacking endosomal TLRs but was unaltered by a lack of TLR2 or MD-2. Together, these findings support the participation of TLR4 and endosomal TLRs in the activation of host macrophages by L. panamensis and in the early control of infection.

Figures

Fig. 1.

L. panamensis infection induces TNF-α production at comparable levels and kinetics to those with specific TLR ligands. (A) Kinetics of TNF-α production in human primary macrophages exposed to different parasite-to-cell ratios, with maximum cytokine secretion at 8 h postinfection. Data are representative of cytokine production by macrophages of 10 individuals and are expressed as means ± standard errors of the means (SEM). (B) Production of TNF-α in human primary macrophages from 10 different donors was measured by ELISA. Cells were stimulated with zymosan, LPS, or poly(I:C), infected for 8 h with promastigotes at a 5:1 parasite-to-macrophage ratio, or left untreated as a control. Data are expressed as means ± standard deviations (SD). *, P < 0.05; **, P < 0.01.

Fig. 2.

L. panamensis induces increased expression of TLRs 1, 2, 3, and 4 in human macrophages. Human macrophages were cultured without treatment, infected with L. panamensis promastigotes at a 5:1 parasite-to-macrophage ratio, or stimulated with zymosan (TLR1), Pam2CSK4 (TLR2), poly(I:C) (TLR3), LPS (TLR4), or CpG (TLR9). TLR expression was evaluated by flow cytometry after 8 h of exposure to ligands or promastigotes. (Top) Mean fluorescence intensities (MFI) of TLR expression from uninfected (empty triangles) and infected (filled circles) macrophages from 12 different individuals. P values are indicated. (Bottom) Representative histograms of TLR expression of uninfected macrophages and macrophages exposed to specific TLR ligands or L. panamensis promastigotes. Cells were gated on the CD14+ population.

Fig. 3.

L. panamensis promastigotes and amastigotes induce TNF-α production in a dose-dependent manner and activate macrophages through TLR-dependent signaling. (A) TNF-α production by WT macrophages exposed to increasing parasite-to-cell ratios, using opsonized promastigotes or amastigotes, specific TLR ligands (hatched bars), or no treatment. (B) MyD88/TRIF−/− cells were infected at a 100:1 parasite-to-macrophage ratio. Data are representative of three independent experiments and are expressed as means ± SEM. *, P < 0.05.

Fig. 4.

MyD88 or TRIF molecules are required for induction of TNF-α production during L. panamensis infection. TNF-α production was measured in MyD88−/− (A) and TRIF−/− (B) macrophages cocultured with opsonized or nonopsonized L. panamensis promastigotes and amastigotes at a 100:1 parasite-to-macrophage ratio, stimulated with specific TLR ligands or Sendai virus (hatched bars), or left untreated. Data are from three independent experiments and are expressed as means ± SEM.

Fig. 5.

Endolysosomal TLRs play a role in the induction of TNF-α production during L. panamensis infection. TNF-α production was measured in UNC93b-deficient macrophages cocultured with L. panamensis promastigotes or amastigotes at a 100:1 parasite-to-macrophage ratio, stimulated with specific intracellular TLR ligands or LPS (hatched bars), or left untreated (medium). Data are from three independent experiments and are expressed as means ± SEM. *, P < 0.05.

Fig. 6.

L. panamensis induces TNF-α production by macrophages through TLR4 but not TLR2. TNF-α production was measured in TLR2−/− (A) and TLR4−/− (B) macrophage cell lines and in differentiated MD-2−/− BMMs (from C57BL/6 mice) (C). BMMs were left untreated (medium) or cocultured with L. panamensis promastigotes and amastigotes at a 100:1 parasite-to-macrophage ratio or were stimulated with specific TLR ligands or LPS alone or in combination with Eritoran (hatched bars). Figures summarize three independent experiments and are expressed as means ± SEM. *, P < 0.05.

Fig. 7.

TLR4 mediates early macrophage leishmanicidal activity. (A) Parasite infection and survival in WT (C57BL/6) and TLR4−/− macrophages infected with L.p-Luc promastigotes. The figure shows the RLU for three independent experiments performed in duplicate. **, P < 0.01 versus WT cells. (B) Confocal microscopy images of WT and TLR4−/− mouse macrophages infected with L.p-GFP promastigotes. Cell membranes were stained with CellMask Deep Red stain. Images show parasites internalized after 30 min of infection. Images of entire slides (right side) show a representative overview of WT and TLR4−/− macrophage infections.

Fig. 8.

Activation of primary bone marrow macrophages by L. panamensis is TLR4 dependent, and signaling is preferentially mediated by MyD88. TNF-α production was measured in differentiated TLR4−/− (A), MyD88−/− (B), TRIF−/− (C), and MyD88/TRIF−/− (D) BMMs (from C57BL/6 mice) left untreated (medium), cocultured with L. panamensis promastigotes at a 100:1 parasite-to-macrophage ratio, or stimulated with specific TLR ligands or Sendai virus (hatched bars). Figures summarize data from three independent experiments and are expressed as means ± SEM. *, P < 0.05.