Temporal regulation of interleukin-12p70 (IL-12p70) and IL-12-related cytokines in splenic dendritic cell subsets during Leishmania donovani infection

Abstract

Dendritic cells (DC) play an essential role in initiating and directing T-cell responses, in part by production of interleukin-12p70 (IL-12p70), IL-23, and IL-27. However, comparative studies on the capacity for cytokine production of DC subsets are rare. Here, we compare splenic CD8alpha+, CD4+, and double-negative (DN) DC, isolated 5 h to 28 days after Leishmania donovani infection, for (i) production of IL-12p70, (ii) accumulation of IL-12/23p40, IL-12p35, IL-23p19, and IL-27p28 mRNAs, and (iii) their capacity to direct CD4+ T-cell differentiation. At 5 h, conventional DC (cDC) accumulated mRNA for IL-12/23p40 (CD8alpha>CD4>DN), IL-23p19 (CD4>CD8alpha>DN), and IL-27p28 (CD8alpha>CD4>DN), in an infection dose-dependent manner. IL-12p70 was restricted to CD8alpha+ cDC, reflecting the subset-specific accumulation of IL-12p35 mRNA. In contrast, cDC from mice infected for 14 to 28 days accumulated little mRNA for IL-12p40 and IL-12p19, though IL-27p28 mRNA remained detectable (CD8alpha>DN>CD4). IL-12p70 secretion by CD8alpha+ cDC was also absent, reflecting deficient IL-12/23p40, rather than IL-12p35, mRNA accumulation. The capacity of CD8alpha+ cDC isolated early after infection to direct Th1 cell differentiation was mediated through IL-12/23p40, whereas this ability in CD4+ and DN cDC was independent of IL-12/23p40 and did not result from overexpression of Delta 4 Notch-like ligand. However, DN cDC produced gamma interferon (IFN-gamma) and also contained a rare population of CD11c(hi) DX5+ IFN-gamma-producing cells. Our data illustrate the extensive diversity in, and temporal regulation of, splenic cDC subsets during infection and suggest caution in interpreting data obtained with unfractionated or minimally purified DC.

FIG. 1.

Purification of splenic cDC subsets. CD11c⁺ DC from five mice were enriched by magnetic activated cell sorting prior to cell sorting. The purity of each cDC subset used in these studies was >99% and is shown in the top graph and in the three bottom graphs. Ssc,. side scatter.

FIG. 2.

L. donovani infection induces maturation of splenic cDC subsets. CD11c^hi DC subsets from naïve (broken line) or 5-h-infected (2 × 10⁸) (solid line) BALB/c mice were examined for changes using a panel of costimulation markers as well as MHC-II. Subsequent to magnetic activated cell sorting enrichment of CD11c⁺ cells, samples were stained with CD11c, CD4, and CD8 in combination with the markers examined above. Histograms were gated on CD11c^hi cells. Black histograms represent isotype controls. Data represent cDC subsets isolated and pooled from five mice per group and are representative of three experiments.

FIG. 3.

L. donovani-activated cDC polarize antigen-specific DO11.10 T-cell differentiation. Sorted DC subsets (1 × 10⁴) from naïve and mice infected with 2 × 10⁷ (LD) or 2 × 10⁸ (HD) amastigotes were cultured with purified naïve DO11.10 T cells (1 × 10⁵/well) in the presence of 0.1 μg/ml OVA peptide. One week later, cells were restimulated with whole spleen cells and 2 μg/ml OVA peptide for 7 h and subsequently stained for IFN-γ and IL-4. Plots were gated on CD4⁺/KJ1-26⁺ cells. Data are representative of three independent experiments. The numbers indicate the percentage of events within the two quadrants.

FIG. 4.

Differential accumulation of IL-12-related subunit mRNAs in cDC subsets. CD11c^hi DC subsets were sorted on the basis of CD4 and CD8α expression from naïve and infected (2 × 10⁷ [LD] or 2 × 10⁸ [HD] parasites/mouse). (A) IL-12/23p40 mRNA, (B) IL-23p19 mRNA, and (C) IL-27p28 mRNA accumulation in CD4⁺, CD8α⁺, and DN DC were examined by real-time RT-PCR. Target genes were normalized against HPRT. Data represent the means plus standard errors of the means (error bars) for three separate experiments.

FIG. 5.

Regulation of Th cell differentiation by IL-12/23p40 and Delta 4 Notch-like ligand. (A) Sorted DC subsets (1 × 10⁴) from naïve C57BL/6 mice and C57BL/6 mice infected with 2 × 10⁷ (LD) or 2 × 10⁸ (HD) amastigotes were cultured with purified naïve OT-II T cells (1 × 10⁵/well) in the presence of 0.1 μg/ml OVA peptide and in the presence of either a control rat immunoglobulin G2a (IgG2a) MAb (left panels) or anti-IL-12/23p40 MAb (right panels). One week later, the cells were restimulated with whole spleen cells from B6.CD45.1 mice and 2 μg/ml OVA peptide for 7 h and subsequently stained for IFN-γ and IL-4. Plots were gated on CD4⁺ CD45.1⁻ cells. The numbers indicate the percentage of events within the two quadrants. (B and C) IL-12/23p40 (B) and Delta 4 Notch-like ligand (C) mRNA accumulation for each cDC subset from infected mice is shown relative to mRNA accumulation in the same subset isolated from naïve mice. Data are derived from one experiment using cells obtained from five mice as cDC donors.

FIG. 6.

Cytokine subunit mRNA accumulation and T-cell differentiating capacity of cDC subsets are altered during the course of infection. (A) Splenic CD11c^hi DC subsets were isolated from naïve and LD-infected mice (2 × 10⁷) at different times (5 h, 24 h, day 14 [d14], and day 28 [d28]). Ex vivo analysis of IL-12/23p40, IL-23p19, and IL-27p28 mRNA accumulation in CD4⁺ (white bars), CD8α⁺ (gray bars), and DN (black bars) DC were examined by real-time RT-PCR. Target genes were normalized against HPRT. (B) Data from panel A are summarized as changes in the accumulation of cytokine mRNA from infected DC subsets compared to their naïve counterpart. (C) Sorted DC subsets from naïve and infected mice (5 h, day 14, and day 28) were cultured with purified naïve DO11.10 T cells in the presence of 0.1 μg/ml OVA peptide. Expanded cells were subsequently restimulated with whole spleen cells and 2 μg/ml OVA peptide and stained for IFN-γ and IL-4. Plots were gated on CD4⁺/KJ1-26⁺. The numbers indicate the percentage of events within the two quadrants. Data are representative of three independent experiments.

FIG. 7.

Lack of bioactive IL-12p70 in cDC from chronically infected mice reflects the absence of IL-12/23p40 rather than IL-12p35 mRNA accumulation. Cytokine production by purified splenic CD11c^hi cell subsets from naïve mice and mice infected for different time periods (5 h or 28 days [d28]) were examined using ELISA to determine secreted IL-12p70 (A) and real-time RT-PCR methods (B and C). (A) cDC subsets were seeded at a concentration of 1 × 10⁶/ml, and samples were collected after 24 h of culture to determine IL-12p70 levels. (B and C) For ex vivo cytokine mRNA expression, relative quantitation analysis of target genes was normalized against HPRT, and each DC subset was calibrated against its naïve counterpart. Data are shown as means plus standard errors of the means (error bars) for the five mice in each group. rE, relative expression; N.D., not detectable.

FIG. 8.

CD11c⁺ DX5⁺ DC are the major producers of IFN-γ. (A) CD11c^hi DC subsets from naïve mice and mice infected with a low dose for 5 h were sorted on the basis of CD4 and CD8 expression, and ex vivo IFN-γ mRNA accumulation in sorted CD4⁺, CD8⁺, and DN DC was examined by real-time RT-PCR. Target genes were normalized against HPRT. Data represent the means plus standard errors of the means (error bars) from three separate experiments. (B) Splenic CD11c^hi cells from mice infected for 5 h with L. donovani were enriched using CD11c microbeads. Enriched CD11c^hi cells were then cultured in brefeldin A (10 μg/ml) for 4 h and surface stained with antibodies for CD11c, CD4, CD8, and CD49b (DX5). Fixed-cell samples were then permeabilized and stained for intracellular IFN-γ. Dot plots were gated on CD11c^hi cells. Quadrant gates were set on isotype controls. The numbers indicate the percentage of events within the three quadrants. This figure shows the results of one experiment that was representative of three independent experiments.