Biology subjects

Owen, J. R.

Publications and source records attributed to Owen, J. R..

3 recordsLinked to original sources

bioRxiv2021

Biphasic effects of IL-27 during Staphylococcus aureus implant-associated osteomyelitis in mice

Interleukin-27 is a pleiotropic cytokine whose reported functions during bacterial infections are debated as an area of active research. To address this, we investigated the role of IL-27 signaling during Staphylococcus aureus osteomyelitis. Clinically, we observed elevated serum IL-27 levels (20-fold higher, p<0.05) in patients with S. aureus osteomyelitis compared to uninfected patients undergoing elective total joint replacement. Remarkably, IL-27 serum levels immediately following septic death were 60-fold higher vs. uninfected patients (p<0.05), suggesting that IL-27 may be a biomarker of end-stage infection and/or cytokine storm. To test this, we hypothesized that IL-27 mediates bacterial clearance during the acute phase of S. aureus osteomyelitis, and subsequently suppresses inflammation to prevent cytokine storm and osteolysis during chronic infection. In mice, we observed that systemic IL-27 delivery by a recombinant adeno-associated viral vector (rAAV-IL-27) ameliorates surgical site soft tissue infection and peri-implant bone loss during the establishment of implant-associated S. aureus osteomyelitis. This effect was not observed in IL-27 receptor knock-out mice, suggesting a direct role of IL-27/IL-27R signaling on immune and bone cell functions. Examination of IL-27-mediated immune responses via transcriptome analyses of infected tibiae demonstrated that IL-27 is a biphasic cytokine with IL-27/IL-27R activating immunostimulatory responses including Th17, IL-2, TLR, and iNOS signaling early, and subsequently suppressing these pathways during chronic infection. Ex vivo confirmation using murine macrophages revealed that IL-27 co-stimulates TLR signaling to increase the production of nitric oxide, and immunomodulatory cytokines such as IL-10, IL-21, IL-31, and TNF-{beta}, but is not a chemokine. Author SummaryStaphylococcus aureus is the most common pathogen in orthopaedic infections, and hard-to-treat (MRSA) strains cause >50% of these infections. Thus, there is an urgent need to develop immunotherapies to treat these life-threatening S. aureus infections. Currently, the role of multifunctional IL-27 on S. aureus osteomyelitis is unknown. In a clinical study, we observed that IL-27 is an important biomarker for identifying S. aureus osteomyelitis patients, and that elevated serum IL-27 levels correlated with adverse clinical outcomes, such as septic death. In our efforts to uncover the underlying mechanisms, we reveal that IL-27 is a biphasic cytokine, activating proinflammatory immune pathways, including Th17 responses, early during acute S. aureus osteomyelitis, and subsequently repressing them during the chronic phase to prevent cytokine storm and bone damage. These results indicate that immune modulation of IL-27/IL-27R signaling could be a viable therapeutic strategy in mitigating S. aureus osteomyelitis.

immunology↗

bioRxiv2020

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology-directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency is low in zygotes, especially for the introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor template, or a HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

bioengineering↗

bioRxiv2020

Evaluation of Mosaicism and Off Target Mutations in CRISPR-Mediated Genome Edited Bovine Embryos

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrates that the use of Cas9 protein, rather than Cas9 mRNA, results in a higher editing efficiency in bovine embryos while lowering the level of mosaicism. However, further optimization must be carried out for the CRISPR/Cas9 system to become feasible for single-step embryo editing in a commercial system.

bioengineering↗