Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity

Abstract

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses and studied the GPI structure-activity relationship. The data show that GPI signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2-lyso-GPIs is comparable with that of the intact GPIs, whereas the activity of Man(3)-GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man(3)-GPIs) and two fatty acids (sn-2-lyso-GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 and NF-kappaB-signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., Krishnegowda, G., and Gowda, D. C. (2004) J. Biol. Chem. 280, 8617-8627). Our data also show that GPIs are degraded by the macrophage surface phospholipases predominantly into inactive species, indicating that the host can regulate GPI activity at least in part by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate immune responses and malaria pathogenesis.

Fig. 1. Measurement of TNF-α produced by human monocytes and mouse macrophages stimulated with P. falciparum GPIs and their derivatives

Human monocytes (panel A) and mouse bone marrow-derived macrophages (panel B) were plated into 96-well microtiter plates (in each case ~2.5 X 10⁴ cells/well). After overnight culturing, the cells were stimulated with the indicated amounts of Man₄-GPIs, Man₃-GPIs, or sn-2 lyso GPIs (either coated on gold particles or solubilized in ethanol, water, 1-propanol (78:20:2, v/v/v). After 48 h, the culture supernatants were collected, and assayed for TNF-α by ELISA. The experiments were performed three times, each in duplicate, and shown are the results of representative experiments. Error bars indicate the range of duplicate wells. The TNF-α production in mouse macrophage cell lines (J774A.1 and RAW264.7 cells) stimulated with the above GPIs was also studied. In both cell type, the results (not shown) were similar to those obtained for mouse bone marrow derived macrophages.

Fig. 2. Assessment of TNF-α produced by macrophages from the wild type, TLR4^−/−, TLR2^−/− and MyD88^−/− mice treated with P. falciparum GPIs and their derivatives

Bone marrow-derived macrophages from the wild type, TLR4^−/−, TLR2^−/−, and MyD88^−/− mice were plated into 96-well microtiter plates (2.5 X 10⁴ cells/well). The cells were cultured overnight, and then stimulated with the indicated amounts of Man₄-GPIs, Man₃-GPI, and sn-2 lyso-GPIs (all coated on gold particles). After 48 h, the cultured supernatants were collected, and TNF-α measured by ELISA. The experiments were performed three times, each in duplicate and shown are the results of representative experiments. Error bars indicate the range of duplicate wells. Panel A, Stimulation with intact parasite GPIs (Man₄-GPIs); panel B, Stimulation with Man₃-GPIs obtained by treatment of Man₄-GPIs with jack bean α-mannosidase; panel C, Stimulation with sn-2 lyso-GPIs obtained by treatment of Man₄-GPIs with phospholipase A₂.

Fig. 3. Analysis of TNF-α production in primary bone marrow cells from wild type, TLR4^−/−, TLR2^−/−, TLR2/4^−/−, and MyD88^−/− mice stimulated with P. falciparum GPIs

Primary bone marrow cells from the wild type, TLR4^−/−, TLR2^−/−, TLR2/4^−/−, MyD88^−/−, mice were plated into 96-well microtiter plates (1 X 10⁶ cells/well) in DMEM medium, 10% FBS, and then stimulated with 200 nM of Man₄GPIs (coated on gold particles) in the presence of monensin. Similar number of cells, treated with 10 ng/ml LPS (TLR4 ligand), and 1 µg/ml Pam₃CSK₄ (TLR2 ligand) both in the presence of monensin, were used as controls. The cells were blocked with anti-Fc receptor antibody, stained with PE-conjugated rat anti-CD11b antibody, fixed, permeabilized, and then stained with FITC-conjugated rat anti-mouse TNF-α. The cells were stained, and analyzed by FACS. The experiments were repeated four times, and data from a representative set is shown.

Fig. 4. Inhibition of P. falciparum GPI-induced TNF-α production in human monocytes by anti-TLR2 and anti-TLR4 monoclonal antibodies

Human monocytes, plated in 96-well microtiter plates (2.5 X 10⁴ cells/well), were incubated with the indicated amounts of anti-TLR2 IgG2 or anti-TLR4 IgG2 monoclonal antibodies in 1640 RPMI medium containing 10% FBS and 1% penicillin/streptomycin. Cells similarly treated with a mouse IgG2 monoclonal antibody against a human tumor-specific glycoprotein (Ref. 46) was used as controls. After 1 h at 37 °C, the cultures were stimulated with 100 nM Man₄-GPIs for 24 h. The culture supernatants were recovered, and the level of TNF-α assayed by ELISA. The experiments were performed two times, each in duplicate, and the average values are plotted. At all concentrations tested, inhibition by anti-TLR2 and anti-TLR4 antibodies was highly significant (p <0.001), whereas inhibition by control antibody was not significant (p >0.05) by ANOVA.

Fig. 5. TLR2/TLR1 and TLR2/TLR6 are differentially recognized by GPIs with three and two fatty acids

HEK cells were plated in 96-well microtiter plates (4 X 10⁴ cells) and transfected with appropriate human TLRs, CD14 genes plus E-selectin-firefly luciferase, β-actin-Renilla luciferase genes as outlined in “Experimental Procedure.” The transfected cells were stimulated with 400 nM GPIs. Transfected cells were also stimulated with Pam3CSK4 and MALP-2 as controls. After 5 h, the cells were washed, lysed and luciferase activity measured. Experiments were repeated three times, each in triplicates, and in each case similar results were obtained. Data from a representative experiment is shown. Error bars indicate the standard deviation of triplicate wells. The results were analyzed by Student’s t test.

Fig. 6. Analysis of downstream MAPKs phosphorylation and IκBα degradation in murine bone marrow macrophages and human monocytes treated with P. falciparum GPIs

Bone marrow-derived macrophages (5 X 10⁵ cells/well) from wild type, TLR2^−/−,TLR4^−/−, and MyD88^−/− mice and human monocytes (2.5 X 10⁵ cells/well) were plated in 24-well microtiter plates. The cells were cultured overnight as described under “Experimental Procedures”, and then stimulated with 200 nM Man₄-GPIs (coated on gold particles). At the indicated time points, the cultured supernatants were removed, and cells were washed and lysed. The cell lysates were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions. The protein bands on gels were transferred onto nitrocellulose membranes, blocked with 5% fat-free dry milk, and probed with antipeptide and phospho-specific antibodies against ERK1/ERK2, p38, and JNK. The membranes were also probed with antibodies against IkBα and β-tubulin. The bound antibodies were detected with HRP-conjugated goat anti-mouse IgGs and chemiluminescence substrate. P, phospho-specific antibodies.

Fig. 7. P. falciparum GPIs exposed to human monocytes or murine macrophages are degraded by cell surface phospholipases

P. falciparum GPIs (0.1 µg + 200,000 cpm of [³H]GlcN-labeled GPIs) were incubated with human monocytes or murine macrophages in serum-free medium. After 48 h, the culture supernatants were extracted with 1-butanol, and the organic layers were washed with water, dried. The aqueous layers were chromatographed on Bio-Gel P-4, and the radioactivity-containing fractions were pooled and dried. The residues from both the organic and aqueous layers were analyzed by HPTLC using CHCl₃, MeOH, H₂O (10:10:2.4, v/v/v), and GPI bands were visualized by fluorography. Panel A, GPIs incubated with human monocytes; panel B, GPIs incubated with murine bone-marrow derived macrophages. In both cases: Lane 1, untreated P. falciparum GPIs and GPI biosynthetic intermediates; lane 2, GPI recovered in organic layer; lane 3, GPIs recovered in aqueous layers. The positions corresponding to the mobility of sn-2 lyso GPIs (the product of phospholipase A₂ on Man₄- and Man₃-GPIs) and GPI glycan core lacking the PI moiety (the product of phospholipase D) are indicated in the right margin. The identities of the GPIs are indicated in the left margin. EtP-M₄Gn-(A)PI, inositol acylated EtN-P-Man₄-GlcN-PI; EtP-M₃Gn-(A)PI, inositol acylated EtN-P-Man₃-GlcN-PI; M₄Gn-(A)PI, inositol acylated Man₄-GlcN-PI; EtP-M₃Gn-(A)PI, inositol acylated Man₃-GlcN-PI. The experiments were also performed with J774A.1 and RAW264.7 murine macrophage cell lines, and the results (not shown) were similar to those of murine bone marrow-derived macrophages.