Genetic profiling of dendritic cells exposed to live- or ultraviolet-irradiated Chlamydia muridarum reveals marked differences in CXC chemokine profiles

Abstract

Chlamydia trachomatis is a major cause of sexually transmitted disease worldwide for which an effective vaccine is being actively pursued. Current vaccine efforts will be aided by elucidating the interaction between Chlamydia and dendritic cells (DCs). Protective immunity appears to develop slowly following natural infection in humans, and early vaccine trials using inactivated C. trachomatis resulted in partial, short-lived protection with possible enhanced inflammatory pathology during re-infection. Thus, immunity following natural infection with live chlamydia may differ fundamentally from immune responses induced by immunization with inactivated chlamydia. We explored this conjecture by studying the response of DCs exposed to either viable or inactivated [ultraviolet (UV) -irradiated] chlamydia elementary bodies (EBs; designated as Live-EB and UV-EB, respectively) using Affymetrix GeneChip microarrays. Thirty-one immunologically characterized genes were differentially expressed by DCs following exposure to Live-EB or UV-EB, including two glutamic acid-leucine-arginine cysteine-X-cysteine (ELR CXC) neutrophil chemoattractant chemokines, Cxcl1 (KC), and Cxcl2 (MIP-2). Up-regulation of these genes by Live-EB as compared to UV-EB was verified by quantitative reverse transcription-polymerase chain reaction and increased chemokine secretion was confirmed by enzyme-linked immunosorbent assay both in vitro and in vivo. Immunofluorescence and fluorescence-activated cell sorter analysis of chlamydia-infected lung tissue confirmed that Live-EB but not UV-EB induced significant DC and neutrophil infiltration during infection. These observations demonstrate that the development of an antichlamydial immune response is dramatically influenced by chlamydial viability. This has implications as to why early inactivated chlamydial vaccines were ineffective and suggests that new vaccine design efforts may benefit from in vitro DC screening for ELR chemokine expression profiles.

Figure 1

Cytokine and chemokine secretion from cultured C57BL/6 BMD DCs: 1 × 10⁶ BMD DCs were seeded into triplicate wells of a 24-well plate and exposed to Live-EB, UV-EB or LPS. Supernatants were harvested at 24 hr postinfection and used for determination of cytokine/chemokine levels by ELISA in duplicate. Data represent the average ± SD of one experiment; three independent experiments were performed and yielded similar results. *Indicates that the fold change between the given experimental condition and the Control condition is significant at P < 0·05 as determined by the Student's t-test; †indicates that the fold change between the Live-EB the UV-EB values is significant at P < 0·05 as determined by the Student's t-test; ‡indicates that the fold change between either the Live-EB or the UV-EB values as compared to the LPS value is significant at P < 0·05 as determined by the Student's t-test.

Figure 2

Cytokine and chemokine secretion in MoPn-infected lung tissue. Groups of six 4- to 6-week-old female C57BL/6 mice were inoculated intranasally with either PBS, 1·5 × 10³ IFU of Live-EB or 7·5 × 10³ IFU of UV-EB. Three days postinfection the animals were killed and the lungs were homogenized for determination of cytokine/chemokine levels by ELISA. Plots and P-values were generated using SigmaPlot.

Figure 3

Immunohistochemistry analysis of infected lung tissue. Groups of two female C57BL/6 mice were infected intranasally with PBS (a,d) or 1500 IFU of either UV-EB (b,e) or Live-EB (c,f) diluted in PBS. Animals were killed and their lungs were isolated on day 3 (a–c) or day 6 (d–f) postinfection and processed for immunohistochemistry staining. Cellular nuclei were stained with DAPI (blue); DCs were stained for CD11c (green), and neutrophils were stained for Gr-1 (red).

Figure 4

FACS analysis of total inflammatory cells. Groups of two female C57/BL6 mice were inoculated intranasally with PBS or 1500 IFU of UV-EB or Live-EB. Animals were killed and their lungs were isolated on day 6 postinfection and total inflammatory cells isolated and stained for FACS. DCs were stained with CD11c-PE and major histocompatibility II-FITC; neutrophils were stained with CD45-PE and Gr-1-FITC. (a) represents one animal from each group from one representative experiment out of three independent trials; (b) represents the data averaged from one entire representative experiment out of three independent trials.