Productive and Penicillin-Stressed Chlamydia pecorum Infection Induces Nuclear Factor Kappa B Activation and Interleukin-6 Secretion In Vitro

Abstract

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

Keywords: Chlamydia pecorum; HeLa cells; chlamydial persistence; interleukin-6; nuclear factor kappa B.

Figure 1

Experimental Design. Cycloheximide was added to culture medium 2 h before infection. Chlamydia was added to culture medium immediately prior to centrifugation or incubation. When centrifugation was omitted, incubation was continued immediately after addition of Chlamydia. When centrifugation was a variable, non-centrifuged samples were placed adjacent to the centrifuge, covered, during the centrifugation. For 2 hpi experiments, medium was not changed; for 24 hpi experiments, medium was changed immediately after centrifugation. Penicillin was added to culture medium after centrifugation. Upon medium change, cycloheximide level was maintained.

Figure 2

Chlamydia pecorum Induces Nuclear Factor Kappa B (NFκB) nuclear translocation and activation early after infection. HeLa cells were pre-exposed (+), or not (−), to 1 (C) or 5 (A,B) μg/mL cycloheximide (CHX) for 2 h, infected using centrifugation with C. pecorum (Cp), C. trachomatis (Ct), or C. suis (Cs) at a multiplicity of infection (MOI) of 1 or 5 and incubated for 2 h. (A) Immunofluorescence (IF) microscopic analysis, in which NFκB p65 was labeled (green), showed C. pecorum-dependent NFκB nuclear translocation (CHX+). (B) Semi-quantitative analysis of NFκB nuclear translocation assayed by IF microscopy, wherein 100 cells per group were scored positive (+), intermediate (=), or negative (−) for NFκB nuclear translocation (see Section Material and Methods), showed that C. pecorum induced the most robust NFκB nuclear translocation of the chlamydial species (MOI 5) evaluated. CHX pre-exposure potentiated Chlamydia-dependent NFκB nuclear translocation. (C) Quantitative analysis of NFκB activation (CHX +) was measured by an Enzyme-linked Immunosorbent Assay (ELISA)-style assay wherein NFκB binding the target DNA consensus sequence, immobilized in a plate, was detected by immune labeling of the bound, active NFκB p65 subunit. C. pecorum infection significantly increased NFκB activation compared to the mock-infected control (n = 3, mean ± standard deviation, ^*p < 0.05).

Figure 3

Chlamydia pecorum-Induced Nuclear Factor Kappa B (NFκB) Activation and Interleukin-6 Secretion are Detectable 24 hpi, Effects not Abolished by Penicillin-Induced Chlamydial Stress. HeLa cells were pre-exposed (A) or not (B,C) to 1 μg/mL cycloheximide (CHX) for 2 h, infected using centrifugation with C. pecorum (Cp) at a multiplicity of infection (MOI) of 1 and incubated until 24 h post infection (hpi) with or without exposure to 1 unit/mL penicillin G (PenG) in the incubation medium. (A) Representative IF micrographs show NFκB p65 (green) and C. pecorum LPS (red) at 1,000x magnification. White and yellow arrows indicate the nuclei of cells with and without NFκB nuclear translocation, respectively. White and yellow asterisks indicate chlamydial inclusions in cells with and without NFκB nuclear translocation, respectively. Scale bar = 10 μm. (B) NFκB activation, specifically of subunit p65, was assayed by an enzyme-linked immunosorbent assay (ELISA)-style assay of whole cell lysates and showed that C. pecorum significantly induced NFκB activation, regardless of PenG-induced chlamydial stress (n = 3, mean ± standard deviation, ^*p < 0.05). (C) Interleukin-6 (IL-6) secretion was assayed by ELISA evaluation of cell culture medium and showed that C. pecorum significantly induced IL-6 secretion, regardless of PenG-induced chlamydial stress (n = 3, mean ± standard deviation, ^*p < 0.05).

Figure 4

Chlamydia-Infected HeLa Lysates Do Not Show Decreased Nuclear Factor Kappa B (NFκB) Levels. HeLa cells were pre-exposed to 1 μg/mL cycloheximide (CHX), or not, for 2 h, infected using centrifugation with C. pecorum (Cp) or C. suis (Cs) at a multiplicity of infection (MOI) of 1 or 5 and incubated until 2 or 24 h post infection (hpi). In addition to mock-infected controls, extra controls with no CHX added and no centrifugation (Con) were included in some experiments. Cells were lysed with a commercially available kit intended to maintain NFκB activity (active lysates) or with a urea lysis protocol reported to deactivate chlamydial protease activity factor (urea lysates). Western blotting was performed on gels loaded with 8 μg lysate protein per lane. After transfer to membranes, total protein staining (bottom image panels) was carried out on membranes prior to probing for NFκB p65 (top image panels). Graphs show NFκB signals normalized to total protein signals and bars of graphs correspond, in order, to the lanes of adjacent images. (A,B) 2 hpi, CHX pre-exposure, MOI 1 (Cp1 or not noted) unless indicated as MOI 5 (Cp5). (C,D) 24 hpi, no CHX, MOI 1. No reduction in NFκB levels was associated with chlamydial infection in any group. Active lysates data shows representative blots from two experiments and urea lysates data shows blots from single, confirmatory experiments.