Late production of CXCL8 in ruminant oro-nasal turbinate cells in response to Chlamydia abortus infection.

Chlamydia abortus is an obligate intracellular bacterium that is an important cause of ovine abortion worldwide. There are reports of abortions in cattle, but these are very rare compared to the reported incidence in sheep. The bacterium is transmitted oro-nasally and can establish a sub-clinical infection until pregnancy, when it can invade the placenta and induce an inflammatory cascade leading to placentitis and abortion. Early host-pathogen interactions could explain differential pathogenesis and subsequent disease outcome in ruminant species. In this study, we assessed the ability of sheep and cattle oro-nasal turbinate cells to sense and respond to C. abortus infection. The cells expressed toll like receptor (TLR) 2, TLR4, nucleotide oligomerization domain (NOD) 1 and NOD-like receptor pyrin domain containing 3 (NLRP3) mRNA. In response to C. abortus infection, both ovine and bovine turbinate cells produce CXCL8 mRNA and protein late in the bacterial developmental cycle, but do not produce IL-1ß or TNF-α. The UV-inactivated bacteria did not elicit a CXCL8 response, suggesting that intracellular multiplication of the bacteria is important for activating the signalling pathways. The production of innate immune cytokines from cattle and sheep turbinate cells in response to C. abortus infection was found to be largely similar.

Infestation of sheep with Psoroptes ovis, the sheep scab mite, results in recruitment of Foxp3(+) T cells into the dermis.

Regulatory T cells (Tregs) play a central role in maintenance of immune homeostasis by controlling harmful immune responses to inappropriate antigens and are thought to play a key role in modulating hypersensitivity reactions. Infestation of sheep with Psoroptes ovis results in a pronounced cutaneous hypersensitivity-type response, which appears to be crucial for mite survival. We hypothesize that (i) Tregs are involved in sheep scab lesions and (ii) Treg responses may crucially affect lesion development and subsequent mite survival. Foxp3 is a key transcription factor required for generation and maintenance of Tregs in rodents and humans, and is the most widely used marker for Tregs in these species. In this study, we sequence ovine foxp3 and show that it exhibits a high degree of homology with foxp3 from other species. Using a validated immunohistochemical staining technique, we demonstrate that infestation of sheep with P. ovis results in an influx of Foxp3(+) T cells into the skin. Future work will investigate the regulatory function of ovine Foxp3(+) T cells and determine whether the quality of the Treg response to P. ovis plays a role in individual susceptibility to the mite.

Relative quantitative kinetics of interferon-gamma and interleukin-10 mRNA and protein production by activated ovine peripheral blood mononuclear cells.

Interferon-gamma (IFN-gamma) and interleukin (IL)-10 are cross-regulatory cytokines capable of driving and controlling the adaptive host immune response. The inter-relationship between IFN-gamma and IL-10 expression has not been defined in sheep despite biological evidence suggesting that they perform similar functions to their orthologues described in other species. To address this, we have developed a quantitative (q)PCR method to assess relative levels of IFN-gamma and IL-10 mRNA expression in activated ovine peripheral blood mononuclear cells (PBMC) and compared the kinetics of mRNA expression with amounts of cytokine secreted by the cells over a 96h period. PBMC were collected from sheep immunised with the nominal antigen ovalbumin (Ova) and re-stimulated in vitro with antigen and the T cell mitogen concanavalin A (ConA). The recall response to antigen was characterised by a single peak in IFN-gamma mRNA expression at 48h of culture (13-fold increase over unstimulated cells) and relatively lower expression of IL-10 mRNA (average 2-3-fold increase over the 96h culture period). Antigen-driven IFN-gamma protein concentration was greatest at the end of the culture period (96h) whereas IL-10 protein level was not elevated above that observed in unstimulated cells. The typical response to ConA was greater for both cytokines, with IFN-gamma mRNA expression peaking at 6h of culture (133-fold increase) then declining rapidly whereas IL-10 mRNA expression peaked at 24h (16-fold increase) and declined more gradually. Despite these differences in the relative kinetics of mRNA expression in mitogen-activated PBMC, the typical pattern of protein expression of the two cytokines was similar. Both showed a gradual rise in protein concentration starting from 12h of culture which was still rising at the end of the culture period (96h). These data demonstrate that the kinetics of mRNA expression for IFN-gamma and IL-10 in activated ovine PBMC do not necessarily correlate with detectable protein in culture.