Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Efferocytosis by Porcine Macrophages and Modulation of Lipid Mediators of Inflammation in vitro: A New Class of Anti‐Microbial with Anti‐inflammatory and Pro‐resolution Properties?

Abstract

Mucosal inflammatory diseases cause great economic losses in the livestock industry. Previous research has shown that some macrolides effectively modulate bacterial‐induced inflammation in animal models. Tylvalosin (TYL) is a new macrolide antibiotic, meant to treat porcine respiratory diseases, in which inflammation is a major component of pathophysiology. The immune modulating effects of tylvalosin are unknown. We hypothesized that TYL may promote the resolution of inflammation in pigs, hence making it an appealing anti‐infective therapeutic.AimsThe aims of the study were to characterize the effects of this macrolide on (1) apoptosis (2) necrosis and reactive oxygen species (ROS) production (3) cytokine, chemokine and lipid mediators of inflammation – leukotriene B4 (LTB−4), prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) (4) phagocytosis and efferocytosis of neutrophils (PMNs) by macrophages.MethodsPorcine blood was collected from the jugular vein for isolation of PMNs and monocytes. Isolated monocytes were grown and matured (verified by esterase staining) over the course of 7 days. Leukocytes were treated with vehicle, TYL, lincomyosin or positive controls: LPS, staurosporine or calcium ionophore. (1) Apoptosis was measured by western blot for cleaved‐caspase 3, TUNEL staining for nicked DNA, and cell death ELISA (2) lactate dehydrogenase (LDH) assay for necrosis detection was used to assess cell death. ROS levels were assessed using bioassays. (3) Multiplex‐bead based assays and qPCR were employed to assess macrophage cytokine and chemokine levels. Reverse phase‐HPLC and liquid chromatography tandem mass spectrometry were used to assess PMN lipid mediator secretions (4) Macrophage phagocytosis and efferocytosis were determined using zymosan (ZYM) phagocytosis assays, TUNEL staining, and myeloperoxidase (MPO) assay.Results(1) Porcine PMN and macrophage apoptosis were increased in cells exposed to TYL in a time and dose dependent manner (P<0.05), but not in the other groups. (2) Tylvalosin had not effect on LDH or ROS levels in macrophages or PMNs. (3) Tylvalosin reduced CXCL8 mRNA transcription and protein secretion in macrophages. IL‐1α but not IL‐1β protein secretion was inhibited. TYL reduced levels of pro‐inflammatory LTB4 and PGE2, while inducing the production of pro‐resolving LXA4 in TYL‐treated porcine PMNs (4) Treated and untreated macrophages had similar levels of phagocytic uptake of ZYM particles. In contrast, MPO revealed a dose dependent increase in efferocytosis of PMN's in macrophages upon TYL treatment (P<0.05).ConclusionsThese findings suggest that TYL promotes apoptosis and efferocytosis of porcine leukocytes, while having no significant effect on necrosis or ROS production. The effect appears to be, at least in part drug‐selective, and does not affect phagocytic function. TYL inhibits LTB4 and PGE‐2, while enhancing the production of LXA4. Together, these results suggest that tylvalosin may have anti‐inflammatory and pro‐resolving properties in porcine tissues.Support or Funding InformationECO Animal Health, Natural Sciences and Engineering Research Council & University of Calgary