Transcriptomic Study on Ovine Immune Responses to <i>Fasciola hepatica</i> Infection

<div><p>Background</p><p><i>Fasciola hepatica</i> 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.</p><p>Methodology/principal findings</p><p>This <i>in vivo</i> study investigated the global gene expression changes of ovine peripheral blood mononuclear cells (PBMC) response to both acute & chronic infection of <i>F</i>. <i>hepatica</i>, and revealed 6490 and 2364 differential expressed genes (DEGS), respectively. Several transcriptional regulators were predicted to be significantly inhibited (e.g. <i>IL12</i> and <i>IL18</i>) or activated (e.g. <i>miR155-5p</i>) 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 ‘<i>IL17</i> 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 <i>F</i>. <i>hepatica</i> infection are a discussed.</p><p>Significance</p><p>In conclusion, the present study performed global transcriptomic analysis of ovine PBMC, the primary innate/adaptive immune cells, in response to infection with <i>F</i>. <i>hepatica</i>, 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.</p></div

Production of nitric oxide in macrophages.

<p>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.</p

PCA and BGA analysis.

<p>(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 <i>F</i>. <i>hepatica</i> 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.</p

Upstream regulator analysis predicts IL18 and IL12 (complex) to be inhibited in PBMC in early infection with <i>F</i>. <i>hepatica</i>.

<p>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).</p

The selected gene expression level in several pathways of interest.

<p>The selected gene expression level in several pathways of interest.</p

Top 10 overlapped IPA canonical pathways in T2vsT1 and T3vsT2 respectively, ranked by overlap p-value.

<p>Top 10 overlapped IPA canonical pathways in T2vsT1 and T3vsT2 respectively, ranked by overlap p-value.</p

Timeline of the experimental vaccination trial.

<p>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.</p

Predicted upstream regulators of interest at acute stage of <i>F</i>. <i>hepatica</i> infection (T2vsT1), ranked by overlap p-value.

<p>Predicted upstream regulators of interest at acute stage of <i>F</i>. <i>hepatica</i> infection (T2vsT1), ranked by overlap p-value.</p

The number of significantly differentially expressed genes at each time point post-<i>M. bovis</i> challenge.

<p>The number of upregulated and downregulated genes in the <i>M. bovis</i>-challenged MDM relative to the control MDM at each time point are shown (adjusted <i>P</i>-value 0.05).</p

Principal component analysis for all individual control and <i>M. bovis</i>-challenged MDM at the 2 hour, 6 hour and 24 hour time points.

<p>Principal component (PC) 1 and PC2 are shown (accounting for 23.31% and 17.05% of the total variation, respectively). PCA was performed using data for all genes whose probes passed the data filtering process with Euclidean distance as the distance metric.</p