Abstract
BackgroundPolymorphonuclear neutrophil (PMN) and eosinophil extracellular trap (ETs) formation has recently been described as an important host effector mechanism against invading pathogens. So far, scarce evidence on metazoan-triggered ET formation has been published. We here describe for the first time Haemonchus contortus-triggered ETs being released by bovine PMN and ovine eosinophils in response to ensheathed and exsheathed third stage larvae (L3).MethodsThe visualization of ETs was achieved by SEM analysis. The identification of classical ETs components was performed via fluorescence microscopy analysis. The effect of larval exsheathment and parasite integrity on ET formation was evaluated via Pico Green®- fluorescence intensities. ETs formation under acidic conditions was assessed by using media of different pH ranges. Parasite entrapment was evaluated microscopically after co-culture of PMN and L3. ET inhibition experiments were performed using inhibitors against NADPH oxidase, NE and MPO. Eosinophil-derived ETs were estimated via fluorescence microscopy analysis.ResultsL3 significantly induced PMN-mediated ETs and significant parasite entrapment through ETs structures was rapidly observed after 60 min of PMN and L3 co-culture. Co-localization studies of PMN-derived extracellular DNA with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite-entrapping structures confirmed the classical characteristics of ETs. Haemonchus contortus-triggered ETs were significantly diminished by NADPH oxidase-, NE- and MPO-inhibition. Interestingly, different forms of ETs, i.e. aggregated (aggETs), spread (sprETs) and diffused (diffETs) ETs, were induced by L3. AggETs and sprETs firmly ensnared larvae in a time dependent manner. Significantly stronger aggETs reactions were detected upon exposure of PMN to ensheathed larvae than to exsheathed ones. Low pH conditions as are present in the abomasum did not block ETosis and led to a moderate decrease of ETs. Eosinophil-ETs were identified extruding DNA via fluorescence staining.ConclusionWe postulate that ETs may limit the establishment of H. contortus within the definitive host by immobilizing the larvae and hampering them from migrating into the site of infection. Consequently, H. contortus-mediated ET formation might have an impact on the outcome of the disease. Finally, besides PMN-triggered ETs, we here present first indications of ETs being released by eosinophils upon H. contortus L3 exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1219-1) contains supplementary material, which is available to authorized users.
Highlights
Polymorphonuclear neutrophil (PMN) and eosinophil extracellular trap (ETs) formation has recently been described as an important host effector mechanism against invading pathogens
Besides PMN-triggered extracellular traps (ETs), we here present first indications of ETs being released by eosinophils upon H. contortus L3 exposure
Within the innate immune response polymorphonuclear neutrophils (PMN; [15]) and eosinophils are considered as fundamental leucocytes forming the first line of defense against metazoan nematodes and the first leucocytes to be recruited to the site of infection [16,17,18,19]
Summary
Polymorphonuclear neutrophil (PMN) and eosinophil extracellular trap (ETs) formation has recently been described as an important host effector mechanism against invading pathogens. We here describe for the first time Haemonchus contortus-triggered ETs being released by bovine PMN and ovine eosinophils in response to ensheathed and exsheathed third stage larvae (L3). Within the innate immune response polymorphonuclear neutrophils (PMN; [15]) and eosinophils are considered as fundamental leucocytes forming the first line of defense against metazoan nematodes and the first leucocytes to be recruited to the site of infection [16,17,18,19]. Alongside phagocytosis and oxidative burst, leucocytes are capable of triggering extracellular traps (ETs) as a novel effector mechanism This results in the cellular release of granule proteins and chromatin upon activation that together form extracellular fibers capable of binding and killing Gram-positive and -negative bacteria and parasites [16, 26]. Only two helminth species, i.e. Schistosoma japonicum [41] and Strongyloides stercolaris [15], have been proven to induce NETs
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