Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells

Abstract

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1β, TNF-α, and IL-8. In this study, the expression of IL-1β, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1β and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1β and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.

Keywords: bacterial infection; pathogen-host interaction (MesH); pathogenicity; virulence.

Fig. 1.

Salmonella Typhimurium and Campylobacter fetus subsp. fetus invasion assays in endometrial cells. A gentamicin protection assay was performed to demonstrate that C. fetus subsp. fetus invades bovine endometrial epithelial cells but does not survive.

Fig. 2.

Intracellular survival expression analysis of Campylobacter fetus subsp. fetus and Salmonella Typhimurium in bovine endometrial epithelial cells. An invasion assay was performed, and RNA was extracted from cells. The cDNA was synthesized using random primers, and a quantitative PCR was performed.

Fig. 3.

Interleukin expression in bovine endometrial epithelial cells challenged with Campylobacter fetus subsp. fetus or Salmonella Typhimurium. Expression was analyzed with a 2^–ΔΔCT and compared to control cells (no infected). A student t-test was performed to ΔCT (CT gene of interest – geometric media CT housekeeping genes) compared against non-treated cells; *p < 0.05.

Fig. 4.

Interleukin expression in bovine endometrial epithelial cells treated with cytochalasin D and challenged with Campylobacter fetus subsp. fetus. Expression was analyzed with a 2^–ΔΔCT and compared to control cells (non-cytochalasin D-treated cells). A student t-test was performed to the ΔCT (CT gene of interest – geometric media CT housekeeping genes) comparing against non-cytochalasin D-treated cells; *p < 0.05.