Expression of genes encoding Th1 cell-activating cytokines and lymphoid homing chemokines by chlamydia-pulsed dendritic cells correlates with protective immunizing efficacy

Abstract

We studied the expression of cytokines, chemokines, and chemokine receptors by the RNase protection assay in chlamydia-pulsed dendritic cells to better understand their potent anti-chlamydial immunizing properties. We found that chlamydia-pulsed dendritic cells express a complex profile of inflammatory and immunomodulatory molecules. These include CCR-7, interleukin-12, and interferon-induced protein 10, molecules that might influence the homing of pulsed dendritic cells to the site of chlamydial infection and the induction of a local protective CD4(+) Th1 cellular immunity.

FIG. 1

Intravenous delivery of DC pulsed with nonviable chlamydiae elicits strong protective immunity against infectious intravaginal challenge. Mice received either no immunization (naive), 150 IFU of C. trachomatis EBs (MoPn strain) delivered intravaginally (infected), 2.5 × 10⁸ heat-inactivated MoPn EBs in 0.2 ml of HBSS delivered i.v. (HK EB), or 5 × 10⁶ DC pulsed with 2.5 × 10⁸ heat-inactivated MoPn EBs (DC + HK EB) delivered in 0.2 ml of HBSS s.c., i.p., and i.v. One week following the booster immunization, all mice were challenged intravaginally with 150 IFU of MoPn EBs in 5 μl of SPG. Protection was assessed by quantifying the chlamydial IFU recovered from cervicovaginal swabs (shown on the x axis). Day 4 postchallenge data are shown here. Triangles represent individual mice.

FIG. 2

Serum antibody titers following infection or immunization with chlamydia-pulsed DC. Sera were collected from naive mice, infected mice (35 days postinfection), and 14 days following two immunizations (administered 14 days apart) with chlamydia-pulsed DC (DC + HK EB) by i.v. and i.p. injection. Chlamydia-specific IgG1 and IgG2a titers were determined by ELISA. Naive mice did not elicit a chlamydia-specific IgG1 or IgG2a response. Infected mice elicited a predominantly IgG2a response. All mice immunized with chlamydia-pulsed DC by i.v. injection elicited a strong chlamydia-specific IgG2a and IgG1 response with higher titers than did mice that had been infected and resolved infection for 35 days. Three of the five mice immunized by i.p. injection elicited a chlamydia-specific IgG2a response. Ovals represent individual mice (solid ovals, IgG2a; open ovals, IgG1). Solid horizontal bars indicate the mean absorbance. Absorbance is shown on the y axis; 1:256 dilution of all sera is indicated on the x axis.

FIG. 3

DC pulsed with chlamydiae differentially express chemokines important for recruitment and activation of T cells and immature DC. RNA was harvested from DC for RPA analysis at 2, 12, 24, and 48 h following no treatment (NT) or treatment with LPS, chlamydiae (heat-inactivated MoPn EBs), or latex beads. Unprotected mRNA probes for selected chemokines are shown in the left-hand lane and named along the y axis. Treatment groups and time points are listed at the top of the gel. L32 and GAPDH are constitutive genes that serve as internal controls. Boxed bands represent differentially expressed genes. ∗, no mRNA detected.

FIG. 4

DC pulsed with chlamydiae differentially express Th1 cytokines. RNA was harvested from DC for RPA analysis at 2, 12, 24, and 48 h following no treatment (NT) or treatment with LPS, chlamydiae (heat-inactivated MoPn EBs), or latex beads. Unprotected mRNA probes for selected cytokines are shown in the left-hand lane in A and B. Treatment groups and time points are listed at the top of the gels. L32 and GAPDH are internal controls. Boxed bands represent differentially expressed genes. ∗, no mRNA detected.

FIG. 5

Expression of CCR7 mRNA by chlamydia-pulsed DC. RNA was harvested from DC for RPA analysis at 2, 24, and 48 h following no treatment (NT) or treatment with heat-inactivated chlamydial EBs or latex beads (LB). Unprotected probes for chemokine receptors 1 to 7 are shown in the left-hand lane. The boxed band indicates increased longevity of expression of the CCR7 mRNA by DC pulsed with nonviable chlamydiae for 48 h.