Prostaglandin E2 modulates dendritic cell function during chlamydial genital infection

Abstract

Inflammatory responses mediated by antigen-presenting dendritic cells (DCs), can be modulated by the presence of prostaglandins (PG), including prostaglandin E2 (PGE2). PGE2 modifies the production of an immune response by altering DC function through PGE2 receptors. PGE2 is produced by epithelial cells lining the murine female reproductive tract during Chlamydia muridarum infection and likely manipulates the antichlamydial immune response during antigen uptake in the genital mucosa. Our data demonstrate that the PGE2 present locally in the genital tract upon chlamydial genital infection enhanced the recruitment of CD11b+ conventional DCs, but not CD45R+ plasmacytoid DCs, to infected genital tract tissue and draining lymph nodes in vivo. Furthermore, exposure to PGE2 in vitro during infection of murine bone-marrow-derived conventional DCs (cDCs) boosted interleukin-10 mRNA and protein while not influencing interleukin-12p40 production. Infection of cDCs markedly increased mRNA production of the costimulatory molecules CD86, CD40 and a member of the C-type lectin family, DEC-205, but addition of PGE2 increased other costimulatory molecules and C-type lectins. Also, exposure of PGE2 to infected cDCs increased FcgammaRIII and FcgammaRIIb, suggesting that PGE2 enhances the uptake and presentation of C. muridarum and augments production of the antichlamydial adaptive immune response. Taken together, the data suggest that exposure of infected cDCs to PGE2 drives production of a diverse adaptive immune response with implications for regulating tissue inflammation.

Figure 1

Prostaglandin E₂ is concentrated in GT tissues upon infection. PGE₂ levels were measured within the serum and GT tissues in MoPn-infected or mock-infected mice on day 8 post infection. Mice were also implanted with a PGE₂ or placebo pellet. (a) PGE₂ levels in serum; (b) PGE₂ levels in GT homogenates. *P < 0·05, compared with placebo + mock infection group by Student's t-test. (c) Ratio of PGE₂ levels in GT homogenates to those in serum. PGE₂ values of individual mice are an average of two independent experiments in (a) and (b). The short horizontal line represents the average level of each group.

Figure 2

Influence of PGE₂ on recruitment of DC subsets during chlamydial infection. DCs were isolated from GT tissue from mice infected with MoPn and implanted with PGE₂ or placebo pellets. A Nycodenz gradient was applied to enrich low-density DCs and cDCs were identified by flow cytometry as described in the Materials and methods section. (a) Total number of DCs, (b) number of conventional or myeloid DCs (cDC), (c) number of plasmacytoid DCs (pDC). The results were obtained from two independent experiments.

Figure 3

Prostaglandin E₂ increases the recruitment of cDC following chlamydial genital infection. Expression of CCR7 on DCs from mice implanted with PGE₂ or placebo pellets was analysed 3 days after a chlamydial infection. Low-density DCs were enriched using a Nycodenz gradient. (a) Representative dot plots of CCR7 expression on CD11c⁺ CD3^– CD19^– cDCs (CD11b⁺) and pDCs (CD11b^–) within GT. (b) The number of DCs expressing high levels of CCR7 within GT were determined 3 days following infection as described in the Materials and methods. (c) The number of DCs within the ILN was determined 3 days following infection as described in the Materials and methods. The results shown in (b) and (c) were obtained from two independent experiments.

Figure 4

Influence of PGE₂ on mRNA expression of selected cytokines and chemokine receptors by infected cDCs. RNA (5–10 μg) was isolated from immature cDCs and infected with MoPn in the presence of PGE₂ or DMSO (control) for 24 hr. SuperArray was performed and normalized to β-actin mRNA internal control. The data are expressed as the ratio of spot intensity of groups as indicated below each panel. A vertical dotted line at the ratio of 1·5 was drawn as the criterion for significant increase in mRNA levels. (a) Ratio of various mRNA species from infected versus mock-infected cDCs treated with DMSO. (b) Ratio of various mRNA species from infected versus mock-infected cDCs treated with PGE₂. (c) Ratio of various mRNA species from infected cDCs treated with PGE₂ versus DMSO. The gene tnf encodes TNF-α. Additional names for genes can be found at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

Figure 5

Quantitative real-time PCR and ELISA analysis of IL-10 and IL-12 cytokine expression by cDCs. Groups of BMDCs (5 × 10⁵) were infected with a multiplicity of infection of 3 live C. muridarum or mock-infected with SPG. PGE₂ or DMSO control (1 μm) was added at time of infection and the cells were cultured. RNA was isolated 30 min after culture and 1 μg RNA was converted to cDNA for quantitative real-time PCR analysis. The results were normalized with the internal control GAPDH. The PCR results were shown as (a) IL-10, (b) IL-12p40. Supernatants were collected from additional wells cultured for 24 hr and cytokine levels were measured by ELISA from undiluted samples and shown as (c) IL-10. Means correspond to the following protein levels as determine with a standard control: infected cDC + PGE₂ = 396 pg/ml; infected cDC + DMSO = 209 pg/ml; mock-infected cDC + PGE₂ = 41 pg/ml and mock-infected cDC + DMSO = 23 pg/ml. The panels are representative of two independent experiments. Statistical comparison of two groups was performed using Student's t-test. Significant P-values are indicated.

Figure 6

Quantification of mRNA expression of genes involved in antigen uptake and antigen presentation by cDCs infected with MoPn. RNA was isolated from cDCs infected with MoPn in the presence of PGE₂ or DMSO control for 24 hr. Between 5 and 10 µg of RNA was used for SuperArray analysis. The data were normalized using the β-actin internal control. The data are expressed as the ratio of spot intensity as indicated in the figure legend. A vertical dotted line at the ratio of 1·5 was drawn as the criterion for significant difference. (a) Ratio of various mRNA species from infected versus mock cDCs treated with DMSO. (b) Ratio of various mRNA species from infected versus mock cDCs treated with PGE₂. (c) Ratio of various mRNA species from infected cDCs treated with PGE₂ versus DMSO. The gene named ly75 is commonly known as DEC-205, Clec4A2 as DCIR (dendritic cell immunoreceptor), Clec4d as MCL (macrophage restricted C-type lectin). Other gene names can be found at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi