Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists

Abstract

Plausible representatives of plasmacytoid dendritic cells (pDCs) in pigs have been characterized as being CD4(hi)CD172(lo). Due to their paucity in blood, we utilized novel fluorescent-activated cell sorting procedures to isolate them from PBMC. The resultant subset was greater than 98% homogeneous in regards to the selected phenotype and contained the preponderance of individuals secreting IFN-alpha after exposure to a known stimulant, transmissible gastroenteritis virus (TGEV). In addition to being a potent source of IFN-alpha, other properties of these porcine CD4(hi)CD172(lo) cells including their morphological transition from a plasma cell-like shape during quiescence to one resembling a dendritic cell (DC) after activation by TGEV and their relatively strong constitutive expression of interferon regulatory factor-7 (IRF-7) conformed to the expectations of genuine pDCs. While a substantial IFN-alpha response was also elicited from the porcine pDCs by pseudorabies virus (PrV), swine influenza virus (SIV), and TLR7 and 9 agonists, there was an agent-dependent induction of varying amounts of IL-2, IL-6, IL-8, IL-12, IFN-gamma, and TNF-alpha. Notably, porcine reproductive and respiratory syndrome virus (PRRSV) failed to provoke the pDCs to secrete any of the measured cytokines except IL-2. Moreover, whereas pDCs exposed to TGEV or the TLR9 agonist rapidly increased IRF-7 production and morphed into DCs with enhanced CD80/86 expression, similar alterations were not observed during incubation with PRRSV. This atypical response of pDCs to PRRSV may contribute to its pathogenesis, which unlike that associated with PrV, SIV or TGEV includes persistent infection and limited development of protective immunity.

Fig. 1

TGEV-mediated induction of IFN-α production by phenotypically distinct porcine PBMC. After depletion of lineage positive (CD2⁺, CD3⁺, and/or CD21⁺) cells by using MACS, the remaining porcine PBMC were separated into CD4^hiCD172^lo or predominately CD4⁻CD172^hi subsets by using a MoFlo^® cell sorter. (A) Histograms of non-fractionated, lineage depleted, and CD4^hiCD172^lo purity-sorted PBMC. The positions and frequencies of CD4^hiCD172^lo cells are shown throughout the purification process while those parameters for the CD4⁻CD172^hi cells are only indicated in the Lineage depleted panel. (B and C) Cumulative and individual IFN-α responses of non-fractionated, lineage positive, CD4⁻CD172^hi, and CD4^hiCD172^lo PBMC to TGEV. An intact PBMC sample and the designated subsets were separately cultured with TGEV for 16 h and then the quantities of secreted IFN-α and frequencies of IFN-α SC were determined by using an ELISA (B) and ELISPOT assay (C), respectively. Bars represent the mean ± SEM of a representative experiment (n = 3).

Fig. 2

Expression of surface markers by the CD4^hiCD172^lo subset of porcine PBMC. A portion of a porcine PBMC sample was stained for only CD4 and CD172 and used to prepare the top panel histogram in which the location of the CD4^hiCD172^lo subset is circled. Electronic gating on this demarcated region in similar histograms generated with other PBMC aliquots that were stained specifically for the indicated surface markers with the respective anti-CD mAb (clear areas) or non-specifically with a corresponding, anti-Ig isotype-matched mAb (shaded areas) prior to treatment with anti-CD4 and CD172 mAbs provided the resultant fluorescence distribution data shown in the lower graphs. Bars define the limits of fluorescent intensity representing a positive signal. Representative histograms (n = 3) are shown.

Fig. 3

Morphology of CD4^hiCD172^lo cells purified from porcine PBMC by using the combined lineage depletion/phenotypic sorting procedure. (A) Light microscope (1000× magnification) image of Diff-Quick^® stained cells. (B) Transmission electron microscopic image of a fixed cell.

Fig. 4

Constitutive expression of IRF-7 by porcine PBMC subsets. Stained porcine PBMC were virtually separated into CD4⁻CD172⁻, CD4⁻CD172^hi, CD4⁺CD172⁻, and CD4^hiCD172^lo subsets in CD4 versus CD172 histograms (comparable to Fig. 1A, PBMC panel). (A) Fluorescent intensities of electronically gated PBMC subsets reacting with anti-IRF-7 polyclonal IgG (clear areas) or a non-specific counterpart (shaded areas). Prior to analysis, areas defining each respective subset in the two histograms were made spatially identical. (B) Mean fluorescence intensities (MFIs) of electronically gated, IRF-7 stained PBMC subsets. Vertical bars represent the mean ± SEM of a representative experiment (n = 3).

Fig. 5

Porcine virus- or TLR agonist-mediated induction of IFN-α production by purified porcine pDCs. Two rounds of selection for the CD4^hiCD172^lo subset of porcine PBMC were performed with a Reflection™ high-speed cell sorter. (A) Histograms of non-fractionated porcine PBMC and of cells remaining after an enrichment or subsequent purity sort for a CD4^hiCD172^lo phenotype. The positions and frequencies of CD4^hiCD172^lo cells are shown throughout the isolation process. (B) Cumulative IFN-α response of purified CD4^hiCD172^lo cells (pDCs) to imiquimod, ODN D19, PrV, PRRSV, SIV, or TGEV. The quantities of IFN-α secreted by purity-sorted pDCs cultured alone (Mock) or with a porcine virus or TLR agonist for 24 h were determined by using an ELISA. Significant differences between the response of mock-treated and virus- or TLR agonist-stimulated pDCs are indicated by an (*p ≤ 0.05) or (**p ≤ 0.01). Bars represent the mean ± SEM of a representative experiment (n = 3).

Fig. 6

Porcine virus- or TLR agonist-mediated induction of pro-inflammatory cytokine production by purified porcine pDCs. Purity-sorted porcine pDCs were cultured alone (Mock) or with imiquimod, ODN D19, PrV, PRRSV, SIV, or TGEV for 24 h and then the quantities of secreted IL-2, IL-6, IL-8, IL-12, TNF-α, and IFN-γ were determined by using the Searchlight chemiluminescent assay. Dotted lines indicate the level of detection (pg/ml) for each cytokine. Significant differences between this value and the quantity of respective cytokine released by virus- or TLR agonist-exposed pDCs are indicated by an (*p ≤ 0.05) or (**p ≤ 0.01). Bars represent the mean ± SEM of a representative experiment (n = 3).

Fig. 7

Porcine virus- or TLR agonist-mediated impact on IRF-7 production by purified porcine pDCs. Purity-sorted porcine pDCs were cultured alone (Mock) or with ODN D19, PRRSV or TGEV for 3 or 5 h. (A) Histograms of IRF-7 stained pDCs previously incubated in the absence (shaded areas) or presence of a virus or TLR9 agonist (solid line, clear areas). The extent of binding of non-specific polyclonal IgG by mock-treated pDCs is indicated by the dotted line images. (B) MFIs of IRF-7 stained pDCs incubated in the absence (Fig. 8A, shaded areas) or presence of a virus or TLR9 agonist (Fig. 8A, solid line, clear areas). Significant differences between the response of mock-treated and virus- or TLR9 agonist-exposed pDCs are indicated by an (*p ≤ 0.05). Bars represent the mean ± SEM of a representative experiment (n = 3).

Fig. 8

Porcine virus- or TLR agonist-mediated induction of the maturation of purified porcine pDCs. Purity-sorted porcine pDCs were cultured alone (Mock) or with ODN D19, PRRSV or TGEV for 24 h. (A) Light microscope (500× magnification) images of Diff-Quick^® stained pDCs. (B) Histograms of CD80/86 stained pDCs that were either freshly purified (shaded areas) or previously incubated in the absence or presence of a virus or TLR9 agonist (solid line, clear areas). Horizontal bars define the limits of fluorescent intensity representing a positive signal. (C) Frequencies of pDCs exhibiting a DC morphology after incubation in the absence or presence of a virus or TLR9 agonist. Diff-Quick^® stained pDCs having an irregular cell surface and dendrites were scored as having matured into DCs. (D) Frequencies of pDCs expressing CD80/86 after incubation in the absence or presence of a virus or TLR9 agonist. Values represent percentages of clear areas designated as being within the positive signal range in histograms of CD80/86 stained pDCs (B). A representative experiment (n = 3) is shown.