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Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in chicken phagocytes
1Animal Science Research, Elanco Animal Health, Greenfield, Indiana 46140, USA.
Abstract:
Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease, and is in development for use in poultry mycoplasma air sacculitis. In order to provide an understanding of clinical efficacy, the in vitro interaction of tilmicosin with three types of chicken phagocytes (MQ-NCSU macrophages, monocyte-macrophages, and heterophils) was evaluated. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the cellular (Cc) to the extracellular (Ce) drug concentration (Cc:Ce). Tilmicosin was avidly accumulated by heterophils (Cc: Ce 138 at 4 h incubation vs 32 and 66, respectively, in MQ-NCSU and monocyte-macrophages) with 61 to 88% localized in the lysosomes. Uptake was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors. However, phagocytosis of Pasteurella multocida and lipopolysaccharide exposure increased tilmicosin uptake by the chicken phagocytes. Upon removal of extracellular tilmicosin, 50% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in antibiotic-free medium, 30% remained cell-associated. Opsonized P. multocida significantly enhanced the release of tilmicosin from all three types of chicken phagocytes. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme, avidin, and beta-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro observations of cellular pharmacology suggest a complex interaction between phagocytes and tilmicosin that contribute to clinical efficacy.
Insights
Tilmicosin, a veterinary antibiotic, is avidly taken up by chicken heterophils and macrophages, enhancing lysosomal enzyme production. This cellular interaction suggests a complex mechanism contributing to its clinical efficacy in poultry.
Area of Science:
- Veterinary Pharmacology
- Cellular Biology
- Immunology
Background:
- Tilmicosin is a semi-synthetic macrolide antibiotic used in veterinary medicine for respiratory diseases in cattle and swine.
- It is under development for treating poultry mycoplasma air sacculitis, necessitating an understanding of its interaction with avian immune cells.
Purpose of the Study:
- To investigate the in vitro interaction of tilmicosin with chicken phagocytes (macrophages and heterophils).
- To elucidate the cellular uptake, localization, and release mechanisms of tilmicosin in these cells.
- To assess the impact of tilmicosin on phagocyte function and lysosomal enzyme production.
Main Methods:
- Radiolabeled tilmicosin was used to quantify uptake by MQ-NCSU macrophages, monocyte-macrophages, and heterophils.
- Uptake was analyzed under various conditions including temperature, pH, and in the presence of metabolic inhibitors.
- Phagocytosis of Pasteurella multocida and lipopolysaccharide exposure were used to modulate uptake.
- Drug efflux and localization within lysosomes were determined.
- Lysosomal enzyme production was measured.
- Neutrophil chemotaxis and bacterial growth inhibition bioassays were employed.
Main Results:
- Tilmicosin was avidly accumulated by heterophils (Cc:Ce ratio of 138 at 4h) compared to macrophages (Cc:Ce ratios of 32 and 66).
- A significant portion (61-88%) of internalized tilmicosin was localized in lysosomes.
- Uptake was dependent on cell viability, temperature, and pH, but not metabolic inhibitors.
- Phagocytosis and lipopolysaccharide exposure enhanced tilmicosin uptake.
- Opsonized P. multocida significantly increased tilmicosin release from phagocytes.
- Tilmicosin uptake stimulated lysosomal enzyme production.
- Neutrophils transported and effluxed bioactive tilmicosin.
Conclusions:
- Chicken phagocytes, particularly heterophils, exhibit high avidity for tilmicosin.
- Tilmicosin's intracellular localization in lysosomes and its influence on lysosomal enzyme production suggest a role in innate immunity.
- The complex cellular pharmacology, including uptake, efflux, and interaction with bacterial components, likely contributes to tilmicosin's clinical efficacy in poultry.