Plasmodium falciparum Activates CD16+ Dendritic Cells to Produce Tumor Necrosis Factor and Interleukin-10 in Subpatent Malaria

Abstract

Background: The malaria causing parasite Plasmodium subverts host immune responses by several strategies including the modulation of dendritic cells (DCs).

Methods: In this study, we show that Plasmodium falciparum skewed CD16+ DC cytokine responses towards interleukin (IL)-10 production in vitro, distinct to the cytokine profile induced by Toll-like receptor ligation. To determine CD16+ DC responsiveness in vivo, we assessed their function after induced P falciparum infection in malaria-naive volunteers.

Results: CD16+ DCs underwent distinctive activation, with increased expression of maturation markers human leukocyte antigen (HLA)-DR and CD86, enhanced tumor necrosis factor (TNF) production, and coproduction of TNF/IL-10. In vitro restimulation with P falciparum further increased IL-10 production. In contrast, during naturally acquired malaria episode, CD16+ DCs showed diminished maturation, suggesting increased parasite burden and previous exposure influence DC subset function.

Conclusions: These findings identify CD16+ DCs as the only DC subset activated during primary blood-stage human Plasmodium infection. As dual cytokine producers, CD16+ DCs contribute to inflammatory as well as regulatory innate immune processes.

Figure 1.

CD16⁺ dendritic cells (DCs) lose CD16 (FcγRIII) expression after short-term culture and Toll-like receptor (TLR)4 stimulation. (A) CD86 expression on circulating blood DC subsets in healthy donors; n = 7 (CD16⁺ DCs and CD1c⁺ DCs), n = 14 (CD141⁺ DCs and pDCs). (B) Representative fresh whole blood gating strategy from a healthy donor to confirm the majority of CD14⁻CD1c⁻HLA-DR^dimCD86⁺ DC are CD16⁺ DC. First, CD14⁺ cells were excluded, then CD16⁺ DCs were identified as lineage (CD3, CD56, and CD19)⁻, HLA-DR⁺, and CD1c⁻. CD1c⁻ DCs were identified as CD86⁻ DCs or CD86⁺ DCs. The CD86⁻ gate comprised plasmacytoid DCs (pDCs), CD141⁺ DCs, and immature CD11c⁻ DCs, with no CD16⁺ DCs identified. (C) CD86 median fluorescence intensity (MFI) on CD1c⁺ DCs, pDCs, and immature DCs in fresh whole blood (circles), after 6-hour culture (triangles) and after 6-hour culture with TLR4 stimulation (squares), n = 6. Box plots show the 10th-90th percentile, median, and interquartile range for data from all participants. (D) The CD86⁺ gate predominantly (median, 93%; IQR, 88%–94%) comprised CD11c⁺CD16⁺ DCs, and there were no CD16⁺ DCs in the CD1c⁻ CD86⁻ DCs gate, n = 6. FSC, forward scatter; SSC, side scatter.

Figure 2.

Cytokine profile of CD16⁺ dendritic cells (DCs) in response to in vitro Toll-like receptor (TLR) stimulation. (A) Representative gating of whole blood nonclassical DCs from a naive donor analyzed for intracellular cytokine production (tumor necrosis factor [TNF], interleukin [IL]-12, and IL-10) at 6 hours of culture after stimulation with TLR4 or without stimulation (NIL). CD16⁺ DCs were identified as CD14⁻, lineage (CD3, CD19, and CD56)⁻, human leukocyte antigen-DR⁺, and CD1c. CD1c⁻ nonclassic DCs were gated according to the CD86 isotype control (IgG2b). Cytokine-secreting cells were gated using the appropriate isotype control. (B) CD16⁺ DC cytokine production in response to TLR1/2 (C) TLR4 or (D) TLR7 stimulation. Toll-like receptor stimulations (dark gray circles) were compared with nonstimulation controls (light gray circles). Mann-Whitney t test was used for comparison between “no-stim” and TLR. Tests were 2-tailed and considered significant if P < .05. FSC, forward scatter; SSC, side scatter.

Figure 3.

Cytokine profile of CD16⁺ dendritic cells (DCs) in response to in vitro Plasmodium falciparum-infected red blood cells (pRBC) stimulation. (A) Representative gating of whole blood nonclassical DCs from a naive donor analyzed for intracellular cytokine production (tumor necrosis factor [TNF], interleukin [IL]-12, and IL-10) at 6 hours of culture after stimulation with unparasitized RBC (uRBC) or pRBC. Gating of nonclassic DCs was performed as per Figure 2A. Mann-Whitney t test was used for comparison between uRBC and pRBC. Tests were 2-tailed and considered significant if P < .05. (B) CD16⁺ DC cytokine production in response to pRBC stimulation. (C) Boolean gating of the absolute proportion of each combination of TNF, IL-12, or IL-10-producing cells after pRBC stimulation. The pRBC stimulations (dark gray circles) were compared with uRBC controls (light gray circles). (D) The relative proportion (or “composition”) of CD16⁺ DC cytokine responders to Toll-like receptor (TLR) and pRBC stimulation. Each slice of pie represents the median of “a Boolean gate”/“total cytokine producing CD16⁺ DC”. Partial permutation tests were used in SPICE version 5.3.

Figure 4.

Plasmodium falciparum infection and stimulation increases interleukin (IL)-10/tumor necrosis factor (TNF) coproduction by CD16⁺ dendritic cells (DCs). (A) Schematic of the induced blood-stage P falciparum malaria (IBSM) clinical trial cohorts, on the days specified (arrows): polymerase chain reaction (PCR), full blood counts, and immunological assays were performed. (B) Parasitemia was determined by quantitative PCR in participants infected with 1800 parasitized red blood cells (pRBC). The dotted line indicates the predetermined parasite treatment threshold of 1000 parasites/mL. Brackets represent the day of antimalarial treatment (Rx) on day 7 (n = 15) or day 8 (n = 24). The mean parasitemia ± the standard error is presented. (C) Longitudinal CD16⁺ DC spontaneous cytokine production and in response to pRBC stimulation during IBSM. Boolean gating of CD16⁺ DCs reveals 7 cytokine-producing populations. Shown are the relative proportion of each combination of TNF-, IL-12-, or IL-10-producing cells with (D) spontaneous cytokine production (No Stim) or in response to pRBC stimulation. Light gray circles represent day 0 (before infection) and dark gray circles represent day 7 (peak parasitemia). The Wilcoxon matched-pairs sign rank test was used to compare longitudinal data. Tests were 2-tailed and considered significant if P < .05. CD16⁺ DC cytokine production was evaluated according to the gating example in Figure 1.

Figure 5.

Induced blood-stage Plasmodium falciparum infection uniquely activates circulating CD16⁺ dendritic cells (DCs). CD16⁺ DCs were assessed longitudinally in volunteers. (A) Fresh whole blood gating strategy for CD16⁺ DCs. Myeloid DC subsets were identified as negative for lineage markers (CD3, CD14, CD19, CD20, CD34, and CD56), HLA-DR⁺ (2nd panel), CD11c⁺CD123⁻ (3rd panel), and CD16⁺ (4th and 5th panels). (B) The absolute number (1st panel) and proportion (2nd panel) of circulating CD16⁺ DCs during induced blood-stage P falciparum malaria (IBSM). (C) HLA-DR (1st panel), CD86 (2nd panel), and FcγRIII (3rd panel) expression on circulating CD16⁺ DCs during IBSM. Box plots show the 10th–90th percentile, median, and interquartile range for data from all participants. Data points outside of box plots represent patients that were outliers. The Wilcoxon matched-pairs signed-rank test was used to compare longitudinal data. Tests were 2-tailed and considered significant if P < .05. FSC, forward scatter; SSC, side scatter.

Figure 6.

Peripheral blood mononuclear cell (PBMC) CD16⁺ dendritic cells (DCs) in adults with clinical Plasmodium falciparum malaria. Patients with uncomplicated P falciparum malaria (UM) were compared with age-matched convalescent adults from the same endemic region who were polymerase chain reaction negative for malaria parasites. (A) HLA-DR, (B) CD86, and (C) CD16 (FcγRIII) expression on CD16⁺ DCs in adults with acute uncomplicated malaria (UM) and at convalescence 28 days after antimalarial treatment. Box plots show the 10th-90th percentile, median, and interquartile range for data from all participants. Mann-Whitney t test was used for comparison between patients with acute infection and patients at convalescence (28 days after antimalarial treatment). MFI, median fluorescence intensity.