Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation

Abstract

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3– NKp46+ cells—a classical natural killer cell phenotype—following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3– NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3– NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets

“…A superior capacity for cytokine production by porcine pDC compared to cDC has been previously demonstrated by others ( 21 ) and by us ( 20 ). To elucidate the role of pDC in cytokine production during helminth-driven responses, we collected supernatants from cultured pDC after 24 hours, and determined concentrations of IL-12p40, TNF-α, IL-4 and IL-10 by ELISA.…”

“…Hence, the depletion of NKp46+ cells from the DC-rich fraction applied in our earlier studies was omitted from the current sorting protocol. Consequently, a substantially higher yield of pDC was obtained compared to our previous work ( 20 ). NK cells were still excluded by gating on CD172a+ cells.…”

“…Porcine bona fide DC subsets were sorted from PBMC according to an adapted version of a previously published protocol ( 20 , 21 ). In brief, CD3ε+, γδ-TCR1+, CD21+ and IgM+ PBMC were first depleted by magnetic cell separation using an autoMACS Pro Separator (Miltenyi Biotec; Bergisch Gladbach NW, Germany; RRID : SCR_018596), followed by a second round of magnetic cell separation to isolate CD14+ monocytes.…”

Evaluation of cytotoxic activity of live toxoplasma gondii tachyzoites and toxoplasma antigen on MCF-7 human breast cancer cell line

“…Heat killed Toxoplasma antigen (TgA) was prepared as previously described [12]. Tachyzoites were suspended in PBS and incubated at 90 °C for15 minutes [12].…”

“…Heat killed Toxoplasma antigen (TgA) was prepared as previously described [12]. Tachyzoites were suspended in PBS and incubated at 90 °C for15 minutes [12]. The antigen was stored in -20 °C for later use and protein concentration was quantified using NanoDrop TM 2000 spectrophotometer (Thermo Scientific, USA).…”