Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9
Citation
Mallikarjunappa, S., Shandilya, U.K., Sharma, A., Lamers, K., Bissonnette, N., Karrow, N.A., Meade, K.G. (2020). Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9. BMC Genetics, [online] 21(1), http://dx.doi.org/10.1186/s12863-020-00925-4
Plain language summary
Johne's disease (JD) is a contagious and incurable disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). Since there are currently no effective vaccines or treatment options available to control JD, genetic selection of potentially resistant individuals is being considered as a complementary strategy to decrease the incidence of JD in Canadian dairy herds. To make a genetic selection, we must first identify in the genes of cows susceptible to MAP infection variations that we want to eliminate from the population. Many variations associated with infection have been reported in the literature. The main objective of this study was to validate these previously identified variations by testing their effect on the offspring of Holstein bulls by estimating the breeding value (EBV) using data obtained from milk, namely by the presence of antibodies against MAP in milk. Although this is an indirect indicator of MAP infection status in cattle, the analysis identified three variations located on chromosomes 16, 23 and 26. Using tools additional genetic (quasi-likelihood analysis and regression analysis), four variations were identified, including two with both approaches and the other two located on chromosomes 9 and 16. In conclusion, the results of this study validate the association of known variations with MAP infection status in the Canadian Holstein breed and underline the need to further study the impact of these variations on gene activity.
Abstract
Background: The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne’s disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate. Results: An IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/− IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-α and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative to unedited cells. Conclusions: The findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.