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

Abstract

BackgroundWuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases.Principal findingsIn vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections.Conclusions/SignificanceHuman innate immune cells are able to recognize, attach to and kill B. malayi microfilariae in an in vitro system. This suggests that, in vivo, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.

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

“…Thus, typical NET characteristics were here confirmed for D. immitis -induced NETs by co-localization experiments on microfilariae and L3-triggered release of extracellular DNA being adorned with histones, NE and MPO. In accordance to studies on other metazoan parasites, L3 and microfilaria-triggered NETosis revealed as time dependent ( 47 – 50 , 56 ). In line with data on B. malayi, Haemonchus contortus, Strongyloides stercoralis , and other metastrongyloid species ( 47 – 50 ), NETs were dissolved via DNase I treatment thereby proving the DNA nature of these structures.…”

“…So far, most studies on parasite-triggered NETosis focused on protozoan parasites ( 38 – 45 ). By contrast, little data exist on metazoan-triggered NET formation ( 46 – 50 ). In the case of filariae, the reports on NETosis come exclusively from Brugia malayi and L. sigmodontis , in which microfilariae/L3 were recently demonstrated as potent triggers of NETs leading to parasite entrapment but not killing in vitro ( 24 , 50 ).…”

“…By contrast, little data exist on metazoan-triggered NET formation ( 46 – 50 ). In the case of filariae, the reports on NETosis come exclusively from Brugia malayi and L. sigmodontis , in which microfilariae/L3 were recently demonstrated as potent triggers of NETs leading to parasite entrapment but not killing in vitro ( 24 , 50 ). It is worth noting that adult D. immitis worms survive for an average of 5 years within the definitive host, releasing microfilariae into the bloodstream ( 51 ).…”

Ovine vital neutrophil extracellular traps bind and impair Haemonchus contortus L3 in a breed‐dependent manner

“…It is important to note, however, that resistance to gastrointestinal nematodes in sheep increases with age, and as such, results obtained utilizing a ewe model of infection may differ compared to those using lambs. In addition, human neutrophils have been shown to bind and effectively kill the microfilaria of Brugia malayi in vitro in a manner similar to what is presented here …”

Characterization of Two Trichinella spiralis Adult-Specific DNase II and Their Capacity to Induce Protective Immunity

“…When Brugia malayi microfilariae were incubated with human polymorphonuclear leukocytes (PMNs), the PMNs released ETs that capture the microfilariae. However, after the DNase was added into the medium, the DNase eliminated the PMN attachment to microfilariae ( McCoy et al, 2017 ). The enzyme DNase II has been detected in the ES and surface proteins of T. spiralis AW, IIL, and ML stages, and it affects the host immune system ( Cui et al, 2013b ; Wang et al, 2014 , 2017 ; Liu et al, 2016a ,b).…”