Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep

Abstract

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host's gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3-8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4⁺CD25⁺ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8-14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4⁺CD25⁺ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Figure 1

Timeline for the 22 day long study. All sheep were temperature measured daily (not shown in figure), hematology were assessed at eleven time points (tp) (0, 2, 3, 4, 6, 8, 10, 14 16, 18 and 21 dpi), samples for RNA-isolation collected on eight tp (0, 2, 3, 4, 8, 10, 14 and 21 dpi), samples for bacterial load on four tp (0, 6, 10 and 14 dpi) and sampling for flow cytometry of PBMC on four tp (0, 3, 10 and 14 dpi).

Figure 2

Temperature, bacterial load and hematological parameters. Data on bacterial load (Genomic equivalents, log₁₀) (A, open pentagon, blue, right Y-axis), temperature (Celsius) (A, closed pentagon, orange, left Y-axis), lymphocyte count (B, five-pointed star, purple) (10⁹ cells/L), neutrophil granulocyte count (B, star, green) (10⁹ cells/L). The different symbols represent median observation for the respective time points, while the coloured areas represent the interquartile range (IQR) for the observations. Black stippled line in (A) describes upper limit for normal rectal temperature in sheep. Green stippled line and purple stippled in (B) describe the limits of neutropenia (0.7 × 10⁹ cells/L) and lymphocytopenia (2.0 × 10⁹ cells/L) in sheep. Data were analyzed with Repeated one way multiple comparison, further with Tukey’s multiple comparison test in GraphPad Prism (CA, USA). Significance p < 0.05, * < 0.05, ** < 0.01, * < 0.001. Control sheep are in Supplementary Fig. S1.

Figure 3

Gene expression profile shown in a volcano plot. All expression profiles were compared with expression profile obtained on day 0, prior to inoculation with A. phagocytophilum. The numbers of downregulated genes are represented in blue figures while upregulated genes are displayed in red, respectively. (A) Day 2. (B) Day 3. (C) Day 4. (D) Day 8. (E) Day 10. (F) Day 14. (G) Day 21.

Figure 4

Biological pathways based on GO-numbers in context with neutropenia and lymphocytopenia observed in sheep. Red colour represents innate immune pathways, purple- general immune pathways, blue-adaptive immune system and green consist of various pathways. High intensity in colour reflects high gene ratio, low intensity (towards white) represents low gene ratio. The difference of intensity can only be compared within each pathway, and not against other pathways. Stippled vertical black lines as well as purple coloured areas on days show which days the sampling have been performed on (3, 4, 8, 10, 14 and 21). 14 pathways are listed with letters as follows; (A) cytokine-mediated signaling pathway, (B) interferon-gamma mediated signaling pathway, (C) regulation of Myd88 dependent toll-like receptor signaling pathway, (E) regulation of cytokine production, (F) response to bacterium, (G) leukocyte chemotaxis, (H) response to IFN-γ, (I) regulation of IFN-α production, (J) Gamma delta T-cell activation, (K) biological regulation, (L) regulation of multicellular organismal development, (M) carboxyl acid transmembrane transport, (N) positive regulation of endothelial cell development.

Figure 5

Biological pathways in GO-terms. Significance is described on a yellow–red colour scale, and the size of the circle reflects how many genes are associated with the respective pathway. (A) Day 3. (B) Day 4. (C) Day 8. (D) Day 10. (E) Day 14. (F) Day 21.

Figure 6

DEGs associated with innate immune responses. Results described in log2 fold change (blue-significantly downregulated, red-significantly upregulated). (A) Cytokine, (B) pattern recognition receptors, (C) complement, (D) interferons, (E) Apoptosis and NADPH, (F) Neutrophil, (G) NK-cells.

Figure 7

DEGS associated with adaptive immune responses. Results described in log2 fold change (blue-significantly downregulated, red-significantly upregulated). (A) T-cell, (B) B-cell, (C) Co-stimulatory signals, (D) Th1, (E) Th2, (F) CD8, (G) Treg. Genes assessed to be very important for regulation of a cell type, have been marked with “Master regulator” and a green circle.

Figure 8

PBMC flowcytometry. Total cell count on four time points (days 0, 3, 10 and 14). (A) CD4⁺ T-cell count (open circle, pink), (B) CD8⁺ T-cell count (open triangle, yellow), (C) CD4⁺CD25⁺ T-cell count (closed circle, light pink), (D) CD8⁺CD25⁺ T-cell count (closed triangle, light yellow. (E) CD4 ⁺CD8⁺ ratio. Data were analyzed with Repeated one way multiple comparison, further with Tukey’s multiple comparison test in GraphPad Prism (CA, USA). Significance p < 0.05, * < 0.05, ** < 0.01, * < 0.001. Respective coloured area represent the IQR for the observations.