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

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.

Intra-articular xenogeneic mesenchymal stem cell-based therapy increases CD4+CD25+ cells in synovial fluid.

Osteoarthritis (OA) is a chronic joint disease afflicting a substantial portion of the world's population with no currently available cure. Mesenchymal stem cell (MSC)-based therapies have been observed to have a mild beneficial effect in OA but the mechanism behind their action remains unclear. This study aimed to identify the lymphocytic response to a xenogeneic human umbilical cord-derived MSC-based cell therapy. A unilateral medial meniscal release model was employed in an ovine model of post-traumatic OA, with the contralateral limb employed as the control. A dose of 1.0 × 107 MSCs was administered to a subset of the OA group as well as to a normal sham-operated group. Synovial fluid was aspirated periodically for 13 weeks for flow cytometry analysis. At the termination of the study the stifle joints were collected and analyzed for potential pathologic changes. Cell therapy induced a transient influx of CD4+ leukocytes; there was a similar significant increase in the proportion of CD4+CD25+ and CD4+CD25hi leukocytes in response to cell therapy, the latter being a subset that may be composed of regulatory T cells. There was no significant effect of the cell therapy treatment on the proportion of synovial fluid-derived CD8+ cells or BAQ44A+ B cells. iNOS expression of intimal lining macrophages was evident but reduced in the cell therapy OA group suggesting macrophage phenotype transformation. There were no inflammatory or histological changes that could be attributed to the cell therapy. Cell therapy induced chemotaxis of CD4+ cells to the joint but these cells were not associated with pathological changes, despite their expression of activation markers (CD25+).