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. 2016 Jan 1;196(1):345-56.
doi: 10.4049/jimmunol.1501558. Epub 2015 Nov 23.

The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion

Affiliations

The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion

Kevin W Tosh et al. J Immunol. .

Abstract

As a major natural host for Toxoplasma gondii, the mouse is widely used for the study of the immune response to this medically important protozoan parasite. However, murine innate recognition of toxoplasma depends on the interaction of parasite profilin with TLR11 and TLR12, two receptors that are functionally absent in humans. This raises the question of how human cells detect and respond to T. gondii. In this study, we show that primary monocytes and dendritic cells from peripheral blood of healthy donors produce IL-12 and other proinflammatory cytokines when exposed to toxoplasma tachyzoites. Cell fractionation studies determined that IL-12 and TNF-α secretion is limited to CD16(+) monocytes and the CD1c(+) subset of dendritic cells. In direct contrast to their murine counterparts, human myeloid cells fail to respond to soluble tachyzoite extracts and instead require contact with live parasites. Importantly, we found that tachyzoite phagocytosis, but not host cell invasion, is required for cytokine induction. Together these findings identify CD16(+) monocytes and CD1c(+) dendritic cells as the major myeloid subsets in human blood-producing innate cytokines in response to T. gondii and demonstrate an unappreciated requirement for phagocytosis of live parasites in that process. This form of pathogen sensing is distinct from that used by mice, possibly reflecting a direct involvement of rodents and not humans in the parasite life cycle.

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Figures

FIGURE 1
FIGURE 1
Primary human monocytes exposed to T. gondii secrete proinflammatory cytokines and trigger IL-12-dependent IFN-γ production by NK cells. (A) Elutriated monocytes (106/ml) from healthy donors (n=49) were infected with RH88 or ME49 tachyzoites (MOI 1:1) or stimulated with LPS (100 ng/ml). Supernatants were collected after incubation for 24 h and p40 IL-12 and TNFα measured by ELISA. Each symbol represents an individual donor and the horizontal lines the mean ± SEM of the values. (B) Primary monocytes from 6 donors were incubated with RH88 or ME49 T. gondii or LPS (as described in A). Bars represent the mean ± SEM of the values obtained for the indicated cytokines measured by Multiplex bead-based assay. (C) IFN-γ production by monocytes (white bars), NK cells (black bars) or co-cultured autologous monocytes and NK cells at a 1:1 ratio (checkered bars) in the presence or absence of RH88 tachyzoites was assayed by ELISA in supernatants collected after 24 h. Bars represent the mean ± SEM of the IFN-γ values obtained for three different donors. (D) Supernatants obtained after 24 h culture of monocytes with medium alone, RH88 tachyzoites (MOI 1:1) or R848 (300 ng/ml) were preincubated with anti-p70 IL-12 (white bars) or an isotype control Ab (black bars) and then added to NK cells (×ばつ105/ml) pre-activated with IL-2 (10 U/ml). Bars represent the mean ± SD of the IFN-γ values in NK cell culture supernatants measured by ELISA. Representative results from one of two donors tested are shown *p< 0.05, ** p < 0.01, *** p < 0.001, nd= not detected.
FIGURE 2
FIGURE 2
Both monocyte-derived DC and blood mDC secrete IL-12 and proinflammatory cytokines in response to T. gondii. (A) Column purified undifferentiated monocytes and donor matched in vitro monocyte-derived macrophages or DC (106/ml) from 4 individuals were stimulated with RH88 tachyzoites (MOI 1:1) or LPS (100 ng/ml). Supernatants were collected at 24 h and p40 IL-12 and TNFα assayed by ELISA. (B) Total blood mDC (CD11c+) were FACSort purified form healthy donors (n=6) and exposed to RH88 tachyzoites or LPS as described in (A). Cytokines were measured in the culture supernatants using a bead-based Multiplex assay. Bars represent the mean ± SEM of values obtained for the group of donors. *p< 0.05, ** p < 0.01, nd= not detected
FIGURE 3
FIGURE 3
CD16+ monocytes and CD1c+ DC are the major subsets producing IL-12 and TNFα. (A) CD16+ CD14low, CD16+ CD14+, and CD16neg CD14+ monocytes or (B) CD1c+CD141neg and CD1cneg141+ DC were FACS purified from 5 donors and cultured at ×ばつ106/ml with T. gondii tachyzoites (MOI 1:1) for 24 h. Cytokines in culture supernatants were measured by ELISA. Bars represent the mean ± SEM of values obtained for individual donors. Statistical significance was determined using a nonparametric 2-way ANOVA. *p< 0.05, ** p < 0.01, ns= not significant, nd= not detected
FIGURE 4
FIGURE 4
Physical contact with live parasites is required for monocyte cytokine production in response to T. gondii. (A) CD14+ column-purified elutriated monocytes (×ばつ106/ml) from 9 donors were exposed to RH88 tachyzoites (MOI 1:1) or stimulated with STAg (10 μg/ml), total tachyzoite Extract (10 μg/ml) or recombinant T. gondii profilin (TgPRF) (1μg/ml). Supernatants were collected at 24 h and p40 IL-12 and TNFα measured by ELISA. (B) Monocytes prepared as in (A) from 5 donors were cultured with tachyzoites of the conditional TgPRF KO RH88 strain in which deletion of profilin is ATc inducible. Cytokines were measured by ELISA in culture supernatants after 24 h. (C) Monocytes from 4 donors prepared as in (A) were cultured for 24 h with either RH88 tachyzoites together (black bars) or separated by a 0.4 μm pore size membrane (grey bars) and cytokines were measured by ELISA. (D) Monocytes from 8 donors prepared as in (A) were cultured with live, irradiated, PFA fixed or heat killed parasites (MOI 1:1) for 24 h. TNFα and p40 IL-12 were measured by ELISA. Bars represent the mean ± SEM of the values obtained for the group of donors. Statistical significance in (A and B) was determined using the Kruskal-Wallis test and in (C) using the Student t test. *p< 0.05, ** p < 0.01, *** p < 0.001, nd= not detected, ns= not significant.
FIGURE 5
FIGURE 5
Parasite invasion is not required for T. gondii induced cytokine production by human monocytes. (A) RH88 tachyzoites were pretreated with the irreversible inhibitors DBAP (100 μM), MYB (3 μM) or the vehicle control, DMSO, prior to culture with CD14+ monocytes. p70 IL-12, p40 IL-12 and TNFα were measured by ELISA in culture supernatants 24 h later. Bars represent the mean ± SEM of the values obtained for 8–9 individual donors (B) GFP expressing RH88 tachyzoites treated with MYB or DMSO were cultured with CD14+ monocytes for 3 h. Representative confocal images of invasion and phagocytosis are shown (magnification 63X) after monocytes were stained for GRA7 and LAMP-1. (C) Frequency of GFP+ monocytes (upper bar graph) after 3 h in culture with GFP+ RH88 assessed by flow cytometry and (D) the percentage of invasion versus phagocytic events (lower pie charts) assessed by confocal microscopy in the same cultures. Results are shown as the mean ± SD of the values obtained from three (C) and two (D) different donors. (E) Representative FACS contour-plots gated on live cells showing intracellular p40 IL-12 staining of column purified monocytes cultured in medium alone, with mCherry-RH88 tachyzoites (MOI 1:1) treated with DMSO or MYB, or stimulated with R848 (300 ng/ml) from one of three donors analyzed. *p< 0.05, **p< 0.01, *** p< 0.001, nd= not detected, ns= not significant.
FIGURE 6
FIGURE 6
Cytokine production from total monocytes is dependent on phagocytosis of live tachyzoites and endosomal acidification. (A) Experimental design used to uncouple phagocytosis from parasite invasion showing four possible outcomes by combining treatment of either monocytes or tachyzoites with the irreversible inhibitor MYB. (B) Photomicrographs of monocytes 24 h after exposure to the corresponding conditions in (A), demonstrating the efficacy of the drug treatments outlined in the experimental design. Arrowheads indicate extracellular tachyzoites and arrows indicate intracellular parasites. (C) Monocytes (106/ml) from 6 donors were preincubated with DMSO or MYB (3 μM) and stimulated with LPS (100 ng/ml) or R848 (300 ng/ml) or cultured at an MOI of 1:1 with RH88 tachyzoites that had been preincubated with either DMSO or MYB. Supernatants were collected after 24 h and TNFα and p40 IL-12 measured by ELISA. Bars represent the mean ± SEM of the values obtained for individual donors. (D–E) Elutriated monocytes (106/ml) were stimulated with RH88 tachyzoites (MOI 1:1), LPS (100 ng/ml) or R848 (300 ng/ml) in the presence or absence of NH4Cl (5 mM) (D) or in the presence or absence of Bafilomycin A1 (3 μM) (E). Supernatants were collected 24 h later and cytokines measured by ELISA. Bars represent the mean ± SD of the ELISA values obtained for one representative donor of 3 in (C) and 2 in (D) tested. The photomicrographs shown confirm that the parasites were able to infect and replicate in the drug treated cultures. Arrows indicate intracellular parasites. *p< 0.05, nd= not detected.
FIGURE 7
FIGURE 7
Phagocytosis of tachyzoites and endosomal acidification are required for the T. gondii-induced cytokine response by both CD16+ monocyte and CD1c+ DC. FACS purified (A) CD16+ NC and IM monocytes or (B) CD1c+ CD141neg DC were pretreated with DMSO (orange bars) or MYB (3μM) (yellow bars) and then cultured (×ばつ106/ml) with DMSO or MYB (3μM) pretreated RH88 (MOI 1:1) or with R848 (300 ng/ml) for 24 h. In parallel, Bafilomycin A1 (3μM) was added for the duration of the culture to the untreated monocytes or DC exposed to MYB-treated parasites (grey bars). Bars represent the mean ± SEM of the values obtained for two donors.

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