Abstract
BackgroundThe 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.ResultsAn 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.ConclusionsThe 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.
Highlights
The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10
Much emphasis has been placed on understanding the genetic basis of susceptibility to Johne’s disease (JD) in cattle [5]; and this has revealed the heritable nature of Mycobacterium avium subsp. paratuberculosis (MAP) infection and led to the identification of single nucleotide polymorphisms (SNPs) across the bovine genome associated with JD phenotypes [6]
The follow-up Western blot analysis revealed that IL10RA protein was undetected in knock out (KO) cell lysate, while it was detected in WT Bovine mammary epithelial cell line (MAC-T) cell lysate (60 kDa protein); thereby validating the generation of IL10RA KO in MAC-T cells (Fig. 2)
Summary
The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. 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. Paratuberculosis (MAP) causes Johne’s disease (JD), a chronic enteritis in cattle. Much emphasis has been placed on understanding the genetic basis of susceptibility to JD in cattle [5]; and this has revealed the heritable nature of MAP infection and led to the identification of single nucleotide polymorphisms (SNPs) across the bovine genome associated with JD phenotypes [6]. While these candidate gene studies reported statistical associations with various phenotypes, they have not confirmed their functional relevance with regards to the host response to MAP infection and/or the pathology of JD
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