Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

Abstract

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.

Trypanosoma brucei brucei Induces Polymorphonuclear Neutrophil Activation and Neutrophil Extracellular Traps Release

“…Different NETs phenotypes were detected in trypomastigoteexposed PMN. These NETs phenotypes have previously been described for other parasite species capable to induce NETs [ (35,36,40); Figure 3 and Supplementary Figures 2, 3]. Correspondingly, diff NETs were identified as a complex of extracellular decondensed chromatin with a size of 15-20 µm diameter, sprNETs consisted of smooth and elongated web-like structures being composed exclusively of thin fibers with a diameter of 15-17 µm and aggNETs were characterized by a "ball of yarn" shape and sizes of more than 20 µm.…”

“…For quantification of different NETs phenotypes [i.e., spread NETs (sprNETs), diffuse NETs (diff NETs), and aggregated NETs (aggNETs)] we followed the description by (35,36). Briefly, bovine PMN (n = 3) were seeded (2 × 10 5 /sample, in duplicates) on 0.01% poly-L -lysine (Sigma-Aldrich) pre-coated coverslips (Thermo Fischer Scientific) and exposed to T. b. brucei trypomastigotes (6 × 10 5 , 1:3 PMN: parasite ratio) for 120 min (37 • C, 5% CO 2 ).…”

“…The phenotypes sprNETs, diff NETs, and aggNETs were visualized by staining extracellular DNA with Sytox Orange R (5 µM, Life Technologies), anti-NE (1:500, Abcam #ab68672), and anti-histones (H1, H2A/H2B, H3, and H4; 1:500, Merck #MAB3422) antibodies as previously described (37,38). For visual quantification, five random power vision pictures were taken from each experimental condition using an inverted IX81 R fluorescence microscope equipped with an XM 10 R digital camera (both Olympus) and analyzed microscopically based on typical morphological characteristics according to Muñoz-Caro et al (36).…”

S100A8/S100A9 deficiency increases neutrophil activation and protective immune responses against invading infective L3 larvae of the filarial nematode Litomosoides sigmodontis

“…This increased neutrophil activation in S100A8/A9 -/mice was further associated with an increased apoptosis. According to the increased neutrophil activation, L3 larval motility was significantly reduced in vitro in the presence of S100A8/A9 -/neutrophils compared to WT neutrophils in a DNA and NET formation-dependent manner, a mechanism that was previously described as neutrophil response to third stage larvae of several helminth species [41,62].…”

Extracellular Traps: An Ancient Weapon of Multiple Kingdoms

“…The study showed that ERK1/2, MAPK, and SOCE pathways were involved in the phenotype, and the parasites were entrapped within the NET structures [74]. Recently, two studies showed that canine PMNs also release extracellular traps in response to Trypanosoma cruzi [77] and Dirofilaria immitis microfilariae, and their L3 stage larvae [78]. Finally, also an overexposure to nickel in the form of nickel nitrate, a commonly used heavy metal material in battery manufacturing, triggered the release of extracellular traps from canine neutrophils [143].…”