Leucocyte-derived extracellular trap formation significantly contributes to Haemonchus contortus larval entrapment

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.

Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release

“…Although NET formation was initially characterized in response to bacteria and protozoa [20, 27], it has also been reported for larger multi-cellular pathogens including fungi and three species of parasitic nematodes [19, 24, 25]. Our initial experiments co-culturing B .…”

“…contortus and S . stercoralis L3 larvae were trapped but not killed by NETs alone [24, 25], though DNase I-mediated extracellular trap destruction prevented human PMN-, macrophage- and autologous serum-mediated killing of S . stercoralis larvae.…”

“…malayi Mf-induced DNA release in the absence of autologous serum (Fig 2E), mirroring the results associated with H . contortus [25] and indicating a role for NADPH oxidases in parasitic nematode driven NETosis. Despite this, DPI appeared to have no significant impact on PMN to Mf attachment (Fig 3A) and obvious NET aggregates could be seen within DPI treated wells.…”

Eosinophils in Helminth Infection: Defenders and Dupes

“…Recent reports show that specific antibodies are capable of cooperating with basophils or alternatively activated macrophages to impede the mobility of N. brasiliensis or Heligmosomoides larvae, respectively [46, 47]. Larvae are also immobilized by eosinophils that are activated to release extracellular traps comprised of DNA fibers and eosinophil granule proteins [48, 49]. These mechanisms merit further investigation.…”

A Trypsin-Sensitive Proteoglycan from the Tapeworm <b><i>Hymenolepis diminuta</i></b> Inhibits Murine Neutrophil Chemotaxis in vitro by Suppressing p38 MAP Kinase Activation

“…This position needs to be re-evaluated. Neutrophils have been implicated in an effective response to the larval stages of the nematodes Lithomosoides sigmodontis, Haemonchus contortus, and Strongyloides steroralis, which can involve myeloperoxidase (MPO) and the release of extracellular traps (i.e., proteases embedded in a chromatin net) [2][3][4][5]. Mice infected with Ascaris have increased numbers of neutrophils in their lungs at the peak of helminth migration (and tissue damage) through the airways [6].…”