Transcriptomic Study on Ovine Immune Responses to Fasciola hepatica Infection

Abstract

Background: Fasciola hepatica is not only responsible for major economic losses in livestock farming, but is also a major food-borne zoonotic agent, with 180 million people being at risk of infection worldwide. This parasite is sophisticated in manipulating the hosts' immune system to benefit its own survival. A better understanding of the mechanisms underpinning this immunomodulation is crucial for the development of control strategies such as vaccines.

Methodology/principal findings: This in vivo study investigated the global gene expression changes of ovine peripheral blood mononuclear cells (PBMC) response to both acute & chronic infection of F. hepatica, and revealed 6490 and 2364 differential expressed genes (DEGS), respectively. Several transcriptional regulators were predicted to be significantly inhibited (e.g. IL12 and IL18) or activated (e.g. miR155-5p) in PBMC during infection. Ingenuity Pathway Analysis highlighted a series of immune-associated pathways involved in the response to infection, including 'Transforming Growth Factor Beta (TGFβ) signaling', 'Production of Nitric Oxide in Macrophages', 'Toll-like Receptor (TLRs) Signaling', 'Death Receptor Signaling' and 'IL17 Signaling'. We hypothesize that activation of pathways relevant to fibrosis in ovine chronic infection, may differ from those seen in cattle. Potential mechanisms behind immunomodulation in F. hepatica infection are a discussed.

Significance: In conclusion, the present study performed global transcriptomic analysis of ovine PBMC, the primary innate/adaptive immune cells, in response to infection with F. hepatica, using deep-sequencing (RNAseq). This dataset provides novel information pertinent to understanding of the pathological processes in fasciolosis, as well as a base from which to further refine development of vaccines.

Fig 1. Timeline of the experimental vaccination trial.

Experimental procedures were carried out under license from the Health Products Regulatory Authority (Project Authorization Number: AE18982/P010) and after ethical review by the University College Dublin (UCD) Animal Ethics Committee.

Fig 2. PCA and BGA analysis.

(A) Principal component analysis (PCA). The 32 samples are projected onto the 2D plane such that they spread out in the two directions that explain most of the differences. The x-axis, so called the first principal component (PC1), is the direction that separates the samples the most. The y-axis, so called the second principal component (PC2), is an unrelated direction (it must be orthogonal to the first direction) that separates the data the second most. The percentage value in the axis label refers to the percent of the total variance that is contained in the direction. Each sample is represented as time points (T0-T3) followed by its animal ID. For example, T3V3 represents the transcriptome sample from vaccinated lamb V3 at time point 3 (14 wpi). (B) The between groups analysis (BGA) plot based on overall gene expression profiles of different groups in response to F. hepatica infection over the period of time. V and C represent vaccinated and control group respectively and their time point (0–3) is separated by a dot in each group.

Fig 3. Venn diagram showing the numbers of DEGS identified from T2vsT1 and T3vsT2.

Overlap comparison of DEGS from two comparisons (T2vsT1 and T3vsT2) according to direction of expression. Sets of up- / down- regulated genes of T2vsT1 are represented in blue and yellow, up- / down- regulated genes of T3vsT2 in pink and green.

Fig 4. TGF-β signaling pathway.

The TGFB signaling pathway is represented with gene expression (log2 fold-change) values overlaid. Red shading indicates increased expression in PBMCs at T2 compared to T1. Green shading indicates decreased expression in PBMC at T2 compared to T1. Color intensity indicates expression level. White and grey shading indicates no significantly differential expression, and filtered out due to low expression respectively.

Fig 5. Production of nitric oxide in macrophages.

This pathway is represented with gene expression (log2 fold-change) values overlaid. Red shading indicates increased expression in PBMCs at T2 compared to T1. Green shading indicates decreased expression in PBMCs at T2 compared to T1. Color intensity indicates expression level. White and grey shading indicates not significantly differentially expressed and filtered out due to low expression, respectively.

Fig 6. Upstream regulator analysis predicts IL18 and IL12 (complex) to be inhibited in PBMC in early infection with F. hepatica.

Downstream target genes are highlighted as upregulated (red) or downregulated (green) at T2vsT1, in the symbols at the edge of the circle, with color intensity increasing with degree of fold change. The activation state of predicted upstream regulators, IL12 (complex) and IL18, is indicated as inhibited (sold blue area within the circle). Arrowheads at the end of interactions (dotted lines) indicate activation, while bars indicate inhibitory effects. The color of lines represent predicted relationships based on gene expression, including orange (activation), blue (inhibition), yellow (findings inconsistent with state of downstream molecule) and grey (effect not predicted).