Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Abstract

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.

Fig. 1.

Type I IFN generated by large low-density (LLD) cells from three different tissues in response to FMDV-immune complexes (IC) and CpG. Shown are results for LLD cells isolated from bovine blood (A), bovine pseudoafferent lymph (B), and bovine mesenteric lymph nodes (C). The results from three individual experiments are shown for each tissue.

Fig. 2.

Sequential enrichment of type I IFN-producing cells by density gradient centrifugation and lineage-specific cell population depletion. (A) FACS analysis of cell populations before (top) and after density gradient centrifugation to enrich for LLD cells (center), followed by depletion of CD3⁺, CD14⁺, and CD21⁺ cells using Miltenyi MACS technology (bottom). (B) Biologically active type I IFN produced by LLD cells at each step of the enrichment after stimulation with FMDV, FMDV-immune complexes (IC), or CpG. Mx-CAT reporter assays showed that enriched populations of cells produced significantly more type I IFN than nonenriched populations (P, <0.001 for total MLN compared to LLD cells; P, 0.0014 for LLD compared to depleted cells) in response to FMDV-immune complexes. The results from three individual experiments are shown for each tissue.

Fig. 3.

Characterization of the cells producing type I interferon in vitro and in vivo. (A) Effects of in vitro depletion of NK cells, γδ T cells, and CD4⁺ cells on the production of type I IFN from cells prepared from mesenteric lymph nodes. Depletion of CD3⁺, CD14⁺, CD21⁺, NK, and γδ T cells significantly enriched type I IFN production in response to FMDV-immune complexes (IC) or CpG [Depleted (+CD4)]. However, depletion of CD4⁺ cells almost completely abolished type I IFN production in response to the same stimuli [Depleted (−CD4)]. (B) Western blot analysis of the level of IRF-7 after stimulation of a cell population depleted of CD3⁺, CD14⁺, CD21⁺, NK, and γδ T cells with immune serum (Ab only), FMDV, FMDV-immune complexes (IC), or CpG. Levels of IRF-7 increased in response to FMDV-immune complexes or CpG but not in response to virus alone, immune serum, or no treatment. Note that IRF-7 could not be detected in LLD cells prior to depletion enrichment. (C) Levels of type I IFN in serum from FMDV-infected cattle depleted of CD4⁺ cells compared to non-CD4⁺-depleted controls (including γδ and CD8-depleted animals). The in vitro depletion studies presented show a positive correlation between the presence of bovine CD4⁺ cells and the production of type I IFN in response to FMDV-immune complexes, comparing large low-density (LLD) cells and CD3⁺, CD14⁺, CD21⁺, NK, and γδ-depleted populations to the CD4⁺-depleted population (P, 0.0019 and 0.013, respectively).

Fig. 4.

Comparison of the level of type I IFN produced by CD11c⁻ and CD11c⁺ cells. (A) FACS analysis of LLD cells depleted of CD3⁺, CD14⁺, and CD21⁺ cells and then separated into CD11c⁻ and CD11c⁺ cell populations. (B) The levels of biologically active type I IFN produced by the CD3⁺, CD14⁺, and CD21⁺ cell-depleted population of LLD cells (Depleted) in response to immune serum (serum), FMDV (virus), FMDV-immune complexes (IC), or CpG were compared to those for the separated CD11c⁻ [Depleted (CD11c⁻)] and CD11c⁺ [Depleted (CD11c⁺)] populations using the Mx-CAT reporter assay. CD11c⁻ cells produced significantly more type I IFN in response to FMDV-immune complexes or CpG (P, 0.0077 and 0.0123, respectively). The results from three individual experiments are shown for each tissue.

Fig. 5.

Phenotype of CD4⁺ CD3⁻ cells capable of expressing type I IFN in response to CpG. (A) FACS analysis of a CD4⁺ CD3⁻ cell population showing the expression of CD172a, MHC II, CD11c, CD45RB, and CD32 by staining. (B) Immunostaining and confocal microscopy carried out on CpG-stimulated cells. (a) pDCs were stimulated for 4 h with CpG in the presence of brefeldin A to trap type I IFN in the ER and were then immunostained for type I IFN. (b) pDCs were stimulated for 16 h with CpG and were then immunostained for type I IFN and CD4. (c) pDCs stimulated for 16 h with CpG were immunostained for CD45RB and CD4 surface expression. (d) pDCs stimulated for 16 h with CpG were immunostained with IgM as an isotype control. (e) pDCs stimulated for 16 h with CpG were immunostained with IgG1 as an isotype control.

Fig. 6.

Type I IFN produced in response to FMDV-immune complexes requires replication-competent/replicating virus. (A) Type I IFN produced by pDC after incubation with either CpG, FMDV (virus), FMDV-immune complexes of live virus (IC), UV-inactivated virus (I-virus), empty capsids (EC), or empty capsids in immune complexes (EC-IC). The results from two individual experiments are shown. (B) Immunostaining of pDCs for FMDV nonstructural protein 3A and CD4. (a) Infected pDCs stained positive for both CD4 and 3A. (Top) DIC image overlays; (bottom) CD4 immunofluorescence (green) and 3A staining (red). (b) Uninfected pDCs stained positive for CD4 but negative for 3A. (Top) DIC image overlay; (bottom) CD4 immunofluorescence (green) and no positive staining for 3A (red).