Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9

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.

Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals

“…In addition, the CRISPR/Cas9 gene editing tool was used to create an IL10RA knock-out in MAC-T cells, which was established from bovine mammary epithelial cells. The findings demonstrate the extensive and critical effects of a knock-out of the IL10RA gene in modifying pro-inflammatory cytokine expression and assisting the immunoregulatory component of IL10RA in inducing an anti-inflammatory response, as well as its possible functional interaction affiliation among immune responses linked to JD [ 129 ].…”

Dectin-1-Mediated Production of Pro-Inflammatory Cytokines Induced by Yeast β-Glucans in Bovine Monocytes

“…Determination of TNF, IL1B, IL6, IL10 and CLEC7A mRNA levels was performed in a CFX96 ™ Real-Time PCR Detection System (Bio-Rad), using NZYSpeedy qPCR Green Master Mix (2×) ROX plus (NZYTech). B2M and MARVEL domain containing 1 (MARVELD1), already used as reference genes in bovine gene expression studies, were used for mRNA normalization (33)(34)(35). Reaction was performed in low profile, non-skirted, 96-well PCR plates (Thermo Fisher Scientific) containing 5 µL Master Mix, 1 µL cDNA, 3.6 µL H2O and 0.2 mM of specific forward and reverse primers (all from Sigma-Aldrich).…”

Johne's Disease in Dairy Cattle: An Immunogenetic Perspective

“…The translation of such techniques is attainable using bovine cell lines that can be further tested to study gene function, gene interaction, and signaling pathway analysis. In the context of MAP infection, gene edited cell lines offer a sound platform to validate biological relevance of JD candidate genes, and related studies are now showing up in the literature (101). Recently, in vitro modeling of intestinal human diseases using 3D-intestinal organoids has gained prominence ( 102), and similar approaches may also be adapted to study JD in cattle.…”

“…The recent evolution of genetic engineering technologies such as CRISPR/cas9 gene editing (179) can be used to validate JD candidate genes. Using the CRISPR/cas9 gene editing technique, we recently created a IL10RA knock-out MAC-T cell line to study functional relevance of candidate gene IL10RA (164) in the context of MAP lysate stimulation (101). IL10RA functions as a trans-membrane receptor of anti-inflammatory cytokine IL-10 known for its immunoregulatory role during JD immune-pathogenesis.…”

“…To date, functional validation of biological relevance of JD candidate genes using CRISPR/cas9 has been limited to gene knock-out studies using the non-homologous end-joining (NHEJ) mechanism (101). However, through this approach, the impact of a risk allele in the context of MAP infection cannot be studied.…”