Impact of Mycobacterium avium subsp. paratuberculosis infection on bovine IL10RA knockout mammary epithelial (MAC-T) cells.

Abstract

Mycobacterium avium subsp. Paratuberculosis (MAP) is an intracellular pathogen that causes Johne's disease (JD) in cattle and other ruminants. IL10RA encodes the alpha chain of the IL-10 receptor that binds the cytokine IL-10, and is one of the candidate genes that have been found to be associated with JD infection status. In this study, a previously developed IL10RA knockout (IL10RAKO) bovine mammary epithelial (MAC-T) cell line and wild-type (WT)MAC-T cells were infected with live MAP for 72h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of IL10RA. Cytokine and chemokine concentrations in culture supernatants were measured by multiplexing immunoassay. Total RNA was extracted from the MAC-T cells, and qPCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of TNF-α, IL-6, CXCL8, CXCL10, CCL2, and CCL3 were significantly induced in WT MAC-T cells and IL-10 was significantly inhibited post-MAP infection. However, IL10RAKO MAC-T cells had greater secretion of TNF-α, IL-6, IFN-γ, CCL3, CCL4, CXCL8, and CXCL10, and lower secretion of VEGF-α. Moreover, the expression of inflammatory genes (TNF-α, IL-1α, IL-6) was also more significantly induced in IL10RAKO cells than in WT MAC-T cells post-MAP-infection, and unlike the WT cells, anti-inflammatory cytokines IL-10 and SOCS3 and chemokines CCL2 were not significantly induced. In addition, the expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP-infection; however, there was no significant induction of these miRNAs in the IL10RAKO cells, which suggests IL10 receptor is somehow involved in regulating the miRNA response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in interleukin signaling, and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of IL10RA in the regulation of innate immune response to MAP.