Lawsonia intracellularis regulates nuclear factor-κB signalling pathway during infection

Abstract

Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) in pigs, horses and wide range of mammals. Little is known about the role of innate immune response during L. intracellularis infection. In this study, we investigated the nuclear factor-κB (NF-κB)-regulated immune response against infection of a clinical strain Dkp23 and a live-attenuated Enterisol vaccine strain in PK-15 cells. We found that expression of NF-κB target genes TNF-α, IFN-γ, IL-6 and IL-8 were modulated during the course of infection. At 5 dpi, there was a significant increase in p65 NF-κB activation, including protein nuclear translocation and phosphorylation, synchronous with the induction of IL-6, IFN-γ and IL-8 expression in L. intracellularis infected cells, especially for Enterisol vaccine strain-infected cells. This result suggests that NF-κB signalling level is induced when L. intracellularis bacterial load peaks at 5 dpi. The induction of pro-inflammatory cytokines expression is consistent with the decreased viability of L. intracellularis-infected cells especially that of the vaccine strain. There were no significant changes in NF-κB signalling between vaccine and Dkp23 infection in PK-15 cells, except for moderate levels of differences in NF-κB target genes expression which might be a reflection of differences in intracellular bacterial load. Overall, the data presented here indicate a correlation between the induction of NF-κB signalling and the L. intracellularis bacterial load in PK-15 cells.

Fig 1. Growth of L. intracellularis strains Dkp23 and Vaccine in PK-15 cell lines.

A qPCR analysis of L. intracellularis genomic copy number in Dkp23 (orange), Vaccine-infected (grey) and uninfected (blue) PK-15 cells at 1, 2, 3, 4 and 5 dpi. Mean values ± standard deviations are shown. Y-axis represents the copy number of L. intracellularis genome. B Immunofluorescence staining of rod-shaped L. intracellularis strains Dkp23 and Vaccine using mouse monoclonal antibody, VPM53, in uninfected and infected PK-15 cells at 1, 3 and 5 dpi, followed by Alexa-488 conjugated secondary antibody (Green). Nuclei were counterstained with DAPI (Blue). White arrows pointed at the infected cells with more than 30 bacteria in cluster. Scale Bar: 10 μm.

Fig 2. Reduced cell viability in Dkp23- and Vaccine-infected PK-15 cells.

Corrected absorbance value of formazan product at 490 nm in uninfected, Dkp23 and Vaccine infected PK-15 cells at 1, 3 and 5 dpi. Mean values ± standard deviations are shown. Y-axis represents the corrected absorbance value at 490 nm.

Fig 3. Regulation of NF-κB target genes mRNA levels in L. intracellularis infected PK-15 cells.

Transcript levels for A) IFN-γ, B) TNF-α, C) IL-6 and D) IL-8 mRNA transcript levels were measured by RT-qPCR at 2 and 8 hpi and 1 3 and 5 dpi with Dkp23 or vaccine strains and data were normalised to the level GAPDH transcript of untreated cells. Mean values ± standard deviations are shown. Y-axis represents fold change in mRNA transcript levels as compared to that of untreated controls.

Fig 4. Regulation of NF-κB nuclear translocation in the nucleus of Dkp23- or Vaccine-infected PK-15 cells.

A Immunofluorescence detection of p65NF-κB and bacteria in Dkp23- (middle panels), Vaccine-infected (bottom panels) and uninfected (upper panels) PK-15 cells at 2 and 8 hpi and 1, 3 and 5 dpi. Alexa-647-conjugated secondary antibody (red) and Alexa-488-conjugated secondary antibody (green) were used to detect p65 NF-κB (p65) and the bacteria (green), respectively. Nuclei were counterstained with DAPI (blue). Scale bars: 10 μm. B Ratio of p65 NF-κB nuclear staining intensity of Dkp23 (orange) and Enterisol (grey) infected PK-15 cells at 2 and 8 hpi and 1, 3 and 5 dpi to that of uninfected controls (blue). Mean values ± standard deviations are shown. Y-axis represent the ratio of p65 NF-κB nuclear staining intensity of infected PK-15 cells to that of uninfected controls. C Representative western blotting images of phosp65 NF-κB, p65 NF-κB and loading control Beta-tubullin. CTL, untreated cells; Dkp23, infection with Dkp23 strain; Vac, infection with the vaccine strain. hpi, hours post infection and dpi, days post infection.

Fig 5. A pulse of TNF-alpha halts the growth of L. intracellularis in-vitro.

A Schematic representation of the experiment. B PK-15 cells were seeded at 30% confluency in 24-wells culture dishes without penicillin/streptomycin supplement and were co-cultured with L. intracellularis (Control, mock infection (MI); DKp23; Vaccine; n = 3) at a MOI of 1 for 1 day. Wells were treated with TNF-α (blue, no TNF-α (non); orange, 25 ng of TNF-α; grey, 50 ng of TNF-α) for 24 h. Then wells were washed with PBS and culture medium. Finally, L. intracellularis bacteria was quantified by qPCR as described in the section of materials and methods on DNA preparation and qPCR analysis of L. intracellularis genomic copy number.