Plasmodium falciparum PfEMP1 Modulates Monocyte/Macrophage Transcription Factor Activation and Cytokine and Chemokine Responses

Abstract

Immunity to Plasmodium falciparum malaria is slow to develop, and it is often asserted that malaria suppresses host immunity, although this is poorly understood and the molecular basis for such activity remains unknown. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a virulence factor that plays a key role in parasite-host interactions. We investigated the immunosuppressive effect of PfEMP1 on monocytes/macrophages, which are central to the antiparasitic innate response. RAW macrophages and human primary monocytes were stimulated with wild-type 3D7 or CS2 parasites or transgenic PfEMP1-null parasites. To study the immunomodulatory effect of PfEMP1, transcription factor activation and cytokine and chemokine responses were measured. The level of activation of NF-κB was significantly lower in macrophages stimulated with parasites that express PfEMP1 at the red blood cell surface membrane than in macrophages stimulated with PfEMP1-null parasites. Modulation of additional transcription factors, including CREB, also occurred, resulting in reduced immune gene expression and decreased tumor necrosis factor (TNF) and interleukin-10 (IL-10) release. Similarly, human monocytes released less IL-1β, IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and TNF specifically in response to VAR2CSA PfEMP1-containing parasites than in response to PfEMP1-null parasites, suggesting that this immune regulation by PfEMP1 is important in naturally occurring infections. These results indicate that PfEMP1 is an immunomodulatory molecule that affects the activation of a range of transcription factors, dampening cytokine and chemokine responses. Therefore, these findings describe a potential molecular basis for immune suppression by P. falciparum.

Keywords: NF-κB; PfEMP1; Plasmodium falciparum; cytokines; immunosuppression; innate immunity; macrophages; malaria; monocytes; transcription factors.

FIG 1

Transgenic parasite strains do not display PfEMP1 on the iRBC surface. (A) CS2-WT iRBCs, CS2-SBP1-KO iRBCs, or uRBCs were treated with 1 mg/ml trypsin or sham treated, and lysates were probed with an antibody against the cytoplasmic acidic terminal segment (ATS) of PfEMP1. Full-length VAR2CSA PfEMP1 is >300 kDa, and cleaved surface PfEMP1 is 90 kDa. The lack of the 90-kDa band represents the absence of PfEMP1 at the surface. Full-length 300-kDa PfEMP1 is still observed, as it represents the large intracellular pool of PfEMP1, which is protected from trypsin degradation. The asterisk indicates antibody cross-reactivity with the human spectrin protein. The Western blot is representative of results from three independent experiments. (B) 3D7-UpsC^R parasites were grown in the presence or absence of WR99210 for 2 weeks, and lysates from uRBCs, 3D7-WT iRBCs, and 3D7-UpsC^R iRBCs with or without WR99210 were probed with an anti-PfEMP1 ATS antibody. The Western blot is representative of results from three independent experiments. (C) Representative plots of CS2-WT and CS2-SBP1-KO cultures incubated with an antibody against the extracellular domain of VAR2CSA PfEMP1 (PAM1.4) and analyzed by flow cytometry, where ethidium bromide-positive cells were identified as iRBCs. NP, no primary antibody (used as a negative control); FSC, forward scatter. (D) Surface PfEMP1 expression levels of CS2-WT, CS2-SBP1-KO, or CS2-PTP1-KO were determined as the proportion of iRBCs that were PAM1.4 positive by flow cytometry. Error bars represent standard deviations of data from duplicates; the graph is representative of results from three independent experiments.

FIG 2

PfEMP1-null parasites induce greater NF-κB activation in macrophages than do wild-type parasites. RAW-ELAM cells were coincubated with uRBCs, CS2-WT iRBCs, CS2-SBP1-KO iRBCs, PBS (vehicle) control, or LPS (3.5 ng/ml; n = 6) (A); uRBCs or trypsin (1 mg/ml)- or sham-treated CS2-WT iRBCs (n = 4) (B); uRBC, CS2-WT iRBCs, or CS2-PTP1-KO iRBCs (n = 3) (C); and uRBCs, 3D7 iRBCs or 3D7-UpsC^R iRBCs (n = 4) (D). Luciferase activity was measured after 12 h, and uRBC background levels were subtracted from iRBC levels. Data were normalized as fold changes over WT values; the LPS level was normalized as the fold change over the value for the vehicle-only control. Data shown are means, and error bars represent standard errors of the means. Ratio-paired t tests were performed, and a P value of <0.05 was considered significant.

FIG 3

CS2-SBP1-KO parasites induce differential activation of a range of transcription factors in macrophages in comparison to CS2-WT parasites. Macrophages were coincubated with CS2-WT or CS2-SBP1-KO iRBCs for 4 h. Nuclear fractions were obtained, and activities of 48 transcription factors were measured by using a transcription factor activation profiling array (n = 3 independent experiments). Data were normalized to the value for the general transcription factor TFIID. Transcription factors found to have a minimum of a 2-fold change in three independent experiments are shown. Data shown are means, and error bars represent standard errors of the means. One-sample t tests were performed, and a P value of <0.05 was considered significant.

FIG 4

CS2-SBP1-KO parasites induce higher expression levels of immune response genes than do CS2-WT parasites. (A) Macrophages were stimulated with CS2-WT iRBCs (circles) or CS2-SBP1-KO iRBCs (squares) for the indicated times. RNA was extracted, and mRNA levels of TNF, IL-10, MIP-1α, NOS2, IL-12α, IL-12β, and IL-18 were measured by using RT-qPCR. Fold changes over the value for the no-stimulus control are shown (n = 4); ratio-paired t tests were performed, and a P value of <0.05 was considered significant. Connecting lines indicate paired CS2-WT- and CS2-SBP1-KO-stimulated samples from the same experimental repeat. (B) Macrophages were stimulated with uRBCs, CS2-WT iRBCs, or CS2-SBP1-KO iRBCs for indicated times; cell culture supernatants were collected; and levels of TNF and IL-10 were measured by an ELISA. Data shown are those after subtraction of the uRBC background (n = 3) and represent means; error bars represents standard errors of the means. Ratio-paired t tests were performed, and a P value of <0.05 was considered significant.

FIG 5

CS2-SBP1-KO parasites induce higher levels of cytokines and chemokines from primary human monocytes than do CS2-WT parasites. (A and B) Monocytes from naive human donors were stimulated for 12 h with uRBCs, CS2-WT iRBCs, and CS2-SBP1-KO iRBCs (n = 11 donors) (A) or uRBCs, 3D7-WT iRBCs, and 3D7-UpsC^R iRBCs (n = 10 donors) (B). Concentrations of cytokines and chemokines in culture supernatants were measured by a multiplex ELISA. Data shown are those after subtraction of the uRBC background, and connecting lines indicate paired WT- and mutant iRBC-stimulated samples. Different donors were used for the data in panels A and B. Paired t tests were performed, with a P value of <0.05 being considered significant. (C) Cells from three donors stimulated as described above for panel A were lifted, attached to slides by cytospinning, fixed, and Field stained. Slides were analyzed by light microscopy, and cells showing phagocytosis of iRBCs or hemozoin were counted. A minimum of 300 cells was counted under each condition; cell numbers were normalized and are displayed as proportions of the total cells counted. Error bars represent standard errors of means. Paired t test showed no significant difference in phagocytosis rates.