Epithelial processed Mycobacterium avium subsp. paratuberculosis induced prolonged Th17 response and suppression of phagocytic maturation in bovine peripheral blood mononuclear cells

Abstract

Johne's disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a chronic, wasting infectious disease in ruminants that causes enormous economic losses to the dairy and beef cattle industries. Understanding the mechanism of persistency of MAP is key to produce novel ideas for the development of new diagnostic methods or prevention techniques. We sought interactions between the host and MAP using epithelial passage model, which mimic initial stage of infection. From the transcriptomic analysis of bovine immune cells (PBMCs), it was suggested that infection through the epithelial cells elicited prolonged Th17-derived immune response, as indicated by upregulation of IL-17A, IL-17F and RORC until 120 h p.i., compared to directly infected PBMCs. Global downregulation of gene expression was observed after 72 h p.i., especially for genes encoding cell surface receptors of phagocytic cells, such as Toll-like receptors and MHC class II molecules. In addition, the cholesterol efflux transporters ABCA1, ABCG1, and APOE, which are regulated by the LXR/RXR pathway, were downregulated. In summary, it would be suggested that the host initiate immune response to activate Th17-derived cytokines, and MAP survives persistently by altering the host adaptive immune response by suppressing surface receptors and manipulating lipid metabolism in phagocytic cells.

Figure 1

Transcriptomic profiling of bovine peripheral mononuclear cells (PBMCs). (A) Experimental design of the analysis. (B) The numbers of differentially expressed genes (DEGs). DEGs of both groups T1 and T2 were obtained when compared to non-infection control of each time point (24 h and 72 h p.i.) based on fold-change  ≥ |2.0| and p-value < 0.05. (C) The numbers of commonly up- and downregulated genes at 24 and 72 h post-infection.

Figure 2

Canonical pathway analysis of DEGs in each experimental group using the IPA tool. (A) Top 20 canonical pathways sorted by p-value. (B) Top 20 canonical pathways sorted by z-score.

Figure 3

Gene expression profile associated with the Th 17 activation pathway. (A) Gene expression profile associated with the Th17 activation pathway annotated by the IPA tool from RNA-seq data. The log₂ Fold-change of each group is described by a color scale. Genes that were not significant (p-value ≥ 0.05 or Log₂FC < 1.0) are shown as N/A. (B) Quantitative PCR (qPCR) analysis of genes associated with Th17 differentiation. Fold-changes of each sample were calculated by 2 − ΔΔCT analysis. Statistical significance was calculated by ANOVA with Tukey’s multiple comparisons test (p-value, *< 0.05; **< 0.01; ***< 0.0005; ****< 0.0001).

Figure 4

Production of IL-17a from bovine peripheral blood mononuclear cells after treatment with MAP. IL-17a in the culture supernatants of PBMCs were quantified with ELISA after infection with MDBK-processed and native MAP. Concentration of IL-17a was measured by absorbance at 450 nm based on standard curve analysis. Statistical significance was calculated by ANOVA with Tukey’s multiple comparisons test (p-value, *< 0.05; **< 0.01; ***< 0.0005; ****< 0.0001).

Figure 5

Ingenuity pathway analyses of the dendritic cell maturation pathway (A) and TREM-1 signaling pathway (B) at 72 h post-infection, and expression level of related genes (C). Genes shown in red indicate upregulation, genes shown in green indicate downregulation, genes shown in orange indicate predicted activation, genes shown in blue indicate predicted inhibition, and an uncolored node indicates that the genes were not differentially expressed in this pathway. The log2 Fold-change of each group is described by a color scale. Genes that were not significant (p-value ≥ 0.05 or Log₂FC < 1.0) are shown as N/A.

Figure 6

Ingenuity pathway analyses of the Liver X receptor/Retinoid X receptor pathway (LXR/RXR pathway) at 72 h post-infection in (A) group T1 and (B) group T2. Genes shown in red indicate upregulation, genes shown in green indicate downregulation, genes shown in orange indicate predicted activation, genes shown in blue indicate predicted inhibition, and an uncolored node indicates that the genes were not differentially expressed in this pathway. (C) Gene expression profile associated with cholesterol transport. The log2 Fold-change of each group is described by a color scale. Genes that were not significant (p-value ≥ 0.05 or Log₂FC < 1.0) are shown as N/A.