Identification of dendritic cell subsets responding to genital infection by Chlamydia muridarum

Abstract

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum. Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naïve T cells in vivo. In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro. However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro. Our findings show that these two DC subsets likely have different functions in vivo. cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.

Figure 1

Identification of DC subsets responding to genital infection by C. muridarum. (A) Shedding of C. muridarum was determined every three days after vaginal inoculation with 1×10⁵ IFU. Data points represent the SEM from 3 mice. (B) FACS plots of DC subsets from the ILN and GT of day 7 infected mice. Plots represent CD11c+CD3−CD19− cells, and are representative of three independent experiments. (C) Phenotyping of pDC was determined as in (B), but were gated on CD11c+CD3−CD19−CD45R+ cells. Black histograms indicate staining for the indicated marker and grey histograms represent isotype controls. For both (B) and (C), tissues from five mice were pooled and treated as described in material and methods.

Figure 2

DC subset distribution and total numbers in the ILN and GT of C. muridarum infected mice. Distribution of DC subsets in the ILN (A) and GT (B) was determined as percentage of DC (CD11c+CD3−CD19−) for cDC (CD11b+ DC) or pDC (CD45R+ DC). Error bars represent the SEM of three independent experiments in which DC subset distribution was determined by pooling of ILNs or GT from five mice. (C) DC subset numbers in naïve (mock) and infected tissues were determined by FACS as in A and B. Naïve mice were inoculated with SPG without C. muridarum (CM). Infected groups represent the combined analysis of DC subsets on day 7 and 10 PI. Data points represent the mean value +/− SEM from three independent experiments in which DC were examined from the pooled tissues of five mice. * p > 0.05 versus uninfected, ** p > 0.05 between DC subsets by Student’s t-test. N.D., not detected.

Figure 3

Localization of DC subsets in the ILN on day 3 PI. ILNs were extracted from mice on day 3 PI, flash frozen, and stored until sectioning as described in the materials and methods. Sections were stained sequentially for T cells (CD3, red), pDC (BST2, green) or cDC (CD11c, green), and nucleii (DAPI, blue). Images represent consecutive 5 um sections examining cDC (A,C,E) or pDC (B,D,F) at 40× magnification objective.

Figure 4

DC subset expression of costimulatory molecules in the ILN. DC subset were examined by FACS as in Figure 1, and further examined for surface expression of costimulatory molecules. Histograms represent gating on cDC (A) or pDC (B) from pooled naive peripheral lymph nodes (grey), day 7 ILN (black), and isotype controls (dotted, open) for expression of the indicated protein. Data are representative of at least three independent experiments, and represent the pooled ILNs of five mice.

Figure 5

pDC reactivity to C. muridarum in vitro. pDC were differentiated from bone marrow precursors and purified as described in materials and methods. (A) Expression of the indicated markers were examined by FACS on pDC as in Figure 4 following 48 hour exposure to live C. muridarum (CM) EBs (black histograms), CpG (hashed histograms), or media alone (grey histograms). Dotted, open histograms were incubated with CM and stained with isotype control for the indicated protein. Data are representative of two independent experiments. (B) Cytokine expression by pDC was determined by ELISA from tissue culture supernatant of the cultures in (A). Data represent the mean values of duplicate measurements from one experiment, and are representative of two independent experiments.