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Ectodomain shedding by ADAM17 (a disintegrin and metalloproteinase 17) in canine neutrophils

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Abstract

ADAM17 is a transmembrane protease expressed by most cells in humans and mice that cleaves cell surface substrates primarily in a cis manner, a process referred to as ectodomain shedding. ADAM17 has numerous substrates and plays a broad role in various physiological processes, including as a key regulator of inflammation. At this time, little is known about ADAM17 expression and function in dogs. A well-established ADAM17 substrate is the leukocyte adhesion protein CD62L (L-selectin). We show that a selective inhibitor of ADAM17, but not an inhibitor of its most closely related family member ADAM10, blocks CD62L shedding upon canine neutrophil activation. We also tested several anti-human ADAM17 monoclonal antibodies (mAbs) for staining canine neutrophils. Although most did not recognize canine neutrophils, the mAbs MEDI3622 and D1(A12) did. They also blocked the downregulation of CD62L upon neutrophil activation. MEDI3622 is a human IgG antibody and we found that a canine chimeric version of this mAb also blocked CD62L shedding by canine leukocytes. Taken together, our findings provide the first direct evidence of ADAM17 expression and sheddase activity in dogs, establishing a potential therapeutic target for various inflammatory disorders.

Introduction

ADAM17 (a disintegrin and metalloproteinase 17) is a transmembrane protease that is constitutively expressed by most cells in the body (Lambrecht et al., 2018). It cleaves its substrates on the cell surface typically in a cis manner at a specific extracellular site, a process referred to as ectodomain shedding. Its catalytic activity is highly inducible upon cell activation by a broad range of stimuli. This process occurs very rapidly and involves ADAM17 transitioning from a low to high activity state through a conformational change and intermolecular interactions (Mishra et al., 2017). ADAM17 substrates have a key role in regulating inflammatory and immune processes and include various cytokines, their receptors, and adhesion molecules (Lambrecht et al., 2018; Mishra et al., 2017; Zunke and Rose-John, 2017). ADAM17 has been well-characterized in human and mouse neutrophils where it directly regulates various effector functions, including their activation, migration, and phagocytosis of antibody-opsonized targets (Mishra et al., 2017). Aberrant ADAM17 induction, however, has pathological consequences (Lambrecht et al., 2018; Mishra et al., 2017; Zunke and Rose-John, 2017). Using models of sepsis, we have reported that mice with ADAM17-null leukocytes have significantly reduced mortality (Long et al., 2012, 2010; Mishra et al., 2016). This was associated with increased neutrophil recruitment, reduced bacteria levels at sites of infection, and markedly reduced systemic levels of proinflammatory cytokines, establishing a central role of ADAM17 in sepsis pathogenesis (Long et al., 2012, 2010; Mishra et al., 2016). Moreover, in human patients, ADAM17 activity and a functional polymorphism of its gene corresponded with sepsis progression (Kermarrec et al., 2005; Shao et al., 2016).

One of the best studied substrates of mouse and human ADAM17 is the leukocyte adhesion protein CD62L (L-selectin) (Ivetic, 2018; Mishra et al., 2017). Canine CD62L has been characterized at the protein, cellular, and functional levels and is very similar to human CD62L (Abbassi et al., 1991; Crockett-Torabi and Fantone, 1997). Similar to human and murine CD62L (Jutila et al., 1990; Kishimoto et al., 1989, 1990), canine CD62L also undergoes a rapid downregulation in expression upon neutrophil activation, suggesting it is similarly regulated by ectodomain shedding (Abbassi et al., 1991). At this time, the function of ADAM17 in canine neutrophils has not been reported. We demonstrate its expression and that blocking its activity disrupts the downregulation of CD62L expression. Given that ADAM17 has a broad immunomodulatory role in humans (Lambrecht et al., 2018; Mishra et al., 2017; Zunke and Rose-John, 2017), this sheddase may be a key therapeutic target in dogs for different inflammatory disorders.

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Animals

Peripheral blood was collected from 15 healthy pet dogs with owner consent. All animals received routine veterinary examinations and vaccines. The dog breeds represented in this study include four mixed breed dogs, three Labrador retrievers, one beagle, one Weimaraner, and one German shorthaired pointer. Breed information was not readily available for five donors due to client de-identification at time of collection. Blood collection was carried out in strict accordance with the recommendations

Effects of selective ADAM17 inhibitors on CD62 L downregulation by activated canine neutrophils

The mAb CL2/6 recognizes canine CD62L and it has been reported that canine neutrophils rapidly downregulate CD62L upon their activation (Abbassi et al., 1991). We observed that neutrophil staining with this mAb rapidly and efficiently decreased upon their treatment with various stimuli, as determined by flow cytometry (Fig. 1A,B). To investigate the involvement of ADAM17 in the downregulation of canine CD62 L, we utilized several small molecule hydroxamate-based metalloproteinase inhibitors.

Declaration of Competing Interest

None.

Acknowledgments

We would like to thank Kathy Stuebner and Amber Winter from the University of Minnesota, College of Veterinary Medicine, Clinical Investigation Center for assistance in acquiring canine peripheral blood samples, Taylor DePauw with flow cytometry assistance, Dr. Jianming Wu for his assistance with the figures and copy editing the manuscript. This work was supported by the National Institutes of Health [grant number HL128580]. KS was supported by a Howard Hughes Medical Institute and Burroughs

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