Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca2+-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid.

Fatty acids have been recognized as regulators of immune function in addition to their known metabolic role. Long-chain fatty acids bind free fatty acid receptor (FFAR)-1/GPR40, which is expressed on bovine neutrophils, and increase responses such as granule release and gene expression. In this study, we investigated the molecular mechanisms governing the up-regulation of cyclooxygenase-2 (COX-2) and IL-8, as well as matrix metalloproteinase (MMP)-9 granule release in FFAR1/GPR40 agonist-stimulated neutrophils. Our results showed that natural (oleic and linoleic acid) and synthetic (GW9508) FFAR1/GPR40 agonists increased ERK1/2, p38 MAPK and Akt phosphorylation, and that the FFAR1/GPR40 antagonist GW1100 reduced these responses. We evaluated the levels of IκBα, a component of the classical activation pathway of the transcription factor NF-κB, and we observed IκBα reduction after stimulation with FFAR1/GPR40 agonists, an effect that was inhibited by GW1100 or the inhibitors UO126, SB203580 or LY294002. FFAR1/GPR40 agonists increased COX-2 and IL-8 expression, which was inhibited by GW1100 and an NF-κB inhibitor. Finally, the FFAR1/GPR40 agonist-induced MMP-9 granule release was reduced by GW1100 and UO126. In conclusion, FFAR1/GPR40 agonists differentially stimulate neutrophil functions; COX-2 and IL-8 are expressed after FFAR1/GPR40 activation via NF-κB, IκBα reduction is FFAR1/GPR40- and PI3K/MAPK-dependent, and MMP-9 granule release is FFAR1/GPR40- and ERK1/2-dependent.

Introducción: La información producida permanentemente por los registros poblacionales de cáncer es el insumo utilizado por los tomadores de decisiones del sistema de salud local y nacional para planificar las estrategias de prevención del cáncer y evaluar el impacto de sus intervenciones. Objetivo: Determinar la incidencia y mortalidad por cáncer en el municipio de Pasto Colombia periodo 2013-2017. Métodos: Estudio observacional descriptivo de la morbimortalidad por cáncer. La recolección y procesamiento de información se realizaron siguiendo recomendaciones del IARC. Las tasas se calcularon según sexo, edad y ubicación del tumor. Resultados: La incidencia global de cáncer fue de 3.759 casos, 1.608 en hombres (Tasa Estandarizada de Edad TAE= 169.4 casos/100,000 hombres-año) y 2.151 casos en mujeres (TAE= 176.6 casos/100,000 mujeres-año). Los tumores más frecuentes en hombres fueron: próstata (25.9%), estómago (16.5%) y pulmón (4.8%) y en mujeres:mama (19.7%), tiroides (12.2%) y cérvix (10.6%). Se presentaron 2.130 muertes por cáncer, 934 en hombres (TAE=97.8 muertes/100,000 hombres-año) y 1.196 muertes en mujeres (TAE=95.1 muertes/100,000 mujeres-año). Las principales causas de mortalidad en hombres fueron los tumores de estómago (24.8%), próstata (12.8%) y pulmón (7.5%). En mujeres: mama (12.2%), estómago (11.6%) y cérvix (10.0%). Conclusiones: El seguimiento quinquenal de los indicadores de carga de cáncer permite realizar comparaciones a nivel nacional e internacional con el fin de ofrecer las bases para planificar y evaluar la implementación de las políticas públicas de salud, relacionadas con la prevención y atención de las causas más comunes de morbimortalidad en Pasto-Colombia.