Characterization of the IL-12 family of cytokines in human dendritic cells infected with live Chlamydia trachomatis

Chlamydia trachomatis (CT) is a clinically important pathogen. In 2–5% of cases, infection results in the development of reactive arthritis. We have previously shown that human dendritic cells (DC) can be readily infected with live CT following in vitro exposure [1]. Infected DC efficiently process chlamydia and present chlamydial antigens to both CD4⁺ [1] and CD8⁺ T cells [1, 2]. Infected DC also produce a number of cytokines including IL-12 and tumour necrosis factor alpha but not IL-10 [1]. The production of IL-12 following infection gives DC the ability to stimulate Th1 responses to chlamydia. Here we have investigated production of another two cytokines from the IL-12 family – IL-23 and IL-27 – by DC infected with live CT. The results were compared with those obtained following DC activation with other inflammatory stimuli.

IL-23 is a heterodimer composed of p19 and p40 subunits, where the p40 subunit is shared between IL-23 and IL-12. IL-27 is a heterodimer composed of p28 and Epstein–Barr virus-induced protein 3 (EBI3) subunits. The main function of both of these cytokines is to stimulate interferon gamma (IFN-γ) production in T cells. However, whereas IL-27 activity primarily affects naïve T cells, IL-23 stimulates both primed and memory cells.

DC were generated in vitro from CD14⁺ monocytes purified from peripheral blood mononuclear cells. After 7 days of culture in the presence of GM-CSF and IL-4, DC were fully differentiated. Immature DC, DC activated with lipopolysaccharide (LPS), IFN-γ heat-killed CT and infected with live CT were used for these experiments. Total RNA was purified using the High Pure RNA isolation kit (Roche). IL-23 oligos used were: p19 forward, 5'-tcg-gca-cga-gaa-caa-ctg-ag-3'; p19 reverse, 5'-tgg-gga-aca-tca-ttt-gta-gtc-t-3'; p40 forward, 5'-att-gag-gtc-atg-gtg-gat-gc-3'; p40 reverse, 5'-att-gct-ggc-att-ttt-gcg-gc-3'. IL-27 oligos used were: p28 forward, 5'-gcg-gaa-tct-cac-ctg-cca-g-3'; p28 reverse, 5'-cgg-gag-gtt-gaa-tcc-tgc-a-3'; EBI3 forward, 5'-cgt-gcc-ttt-cat-aac-aga-gca-3'; EBI3 reverse, 5'-gac-gta-gta-cct-ggc-tcg-g-3'. GAPDH was used as control. PCR settings: 95°C for 3 min followed by 29 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 90 sec. PCR products were resolved on 1.5% agarose gel.

We showed that infection of DC with live CT resulted in the upregulation of message for all four subunits studied (p19, p40, p28, EBI3). The message was detected at 18 and 24 hours post infection. LPS-activated DC upregulated p28, p40 and EBI3 but not p19. There was no p19 message at either 18 or 24 hours post stimulation. The main subunit upregulated following IFN-γ activation was p28 and there was no detectable p19 mRNA at any of the times studied. Interestingly, there was also no expression of p19 when immature DC were exposed to heat-killed CT. Exposure of DC to heat-killed CT did, however, upregulate p28, p40 and EBI3. A small amount of EBI3 message was detected in immature DC, but on none of the other subunits studied.

Whereas activation of DC with LPS, IFN-γ and heat-killed CT stimulates production of IL-27, these stimuli are insufficient to induce p19 upregulation, and in consequence IL-23 production. However, infection of DC with live CT resulted in the production of mRNA for both IL-23 and IL-27. This study highlights important differences in immune responses following exposure to live and heat-killed CT.

Affiliations

1. University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, Cambridge, UK
   • MK Matyszak
   • , JC Goodall
   •  & JSH Gaston

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