Hookworm infections: Reappraising the evidence for a role of neutrophils in light of NETosis

Abstract

In Hookworm infection, neutrophils have long had the image of the villain, being recruited to the site of larval migration because of damage but participating themselves in tissue injury. With recent developments in neutrophil biology, there is an increasing body of evidence for the role of neutrophils as effector cells in hookworm immunity. In particular, their ability to release extracellular traps, or neutrophil extracellular traps (NETs), confer neutrophils a larvicidal activity. Here, we review recent evidence in this nascent field and discuss the avenue for future research on NETs/hookworm interactions.

Keywords: NETosis; helminth; hookworms; neutrophil extracellular traps; neutrophils.

FIGURE 1

Hookworms actively evade NETosis. The non‐activated infectious larvae of hookworm are trapped by neutrophil extracellular traps (NETs) released by neutrophils isolated from human blood. During the transition to parasitism, heat‐activation causes hookworms to secrete anti‐NETs evasion molecules. Three mechanisms of evasion are illustrated: (i) a DNase‐II capable of degrading NETs to evade trapping and cuticle damage, demonstrated in Necator brasiliensis and N. americanus and (ii) a Kunitz‐type Inhibitor (Ace‐KI1) identified in Ancylostoma ceylanicum is proposed to block the formation of NETs by inhibiting NE activity, (iii) an unidentified blocker of TRMP‐2 inhibits oxidative stress‐induced NETs formation. This mechanism has been demonstrated in the cestode Mesocestoides corti. Fluorescent images were obtained by co‐culture of circulatory human neutrophils with N. americanus L3 for 3 h. Activated larvae were placed at 37°C for one night before co‐culture to stimulate ES release. NETs are stained using sytox green and are represented with the LUT fire in Fiji. The figure has been made using Biorender

FIGURE 2

Neutrophil extracellular traps kill larvae via a variety of mechanisms. Neutrophil extracellular traps (NETs) are induced in response to hookworm and related helminth larvae. (A) The mechanism of NETs induction is not yet characterized. The current hypothesis include parasite‐specific products such as glycans and excretory/secretory (ES) products, the multicellular size of larvae, recognition of microbiome/soil‐derived microbial signatures via toll‐like receptors (TLR), or indirect activation from as‐yet‐unknown immune or non‐immune cells. (B) Following hookworm detection, NETosis induction requires NADPH oxidase (Nox), myeloperoxidase (MPO), neutrophil elastase (NE), and peptidylarginine deiminase 4 (PAD4). While neutrophils have been the primary study of hookworm‐induced NETosis, emerging evidence suggests other cells may form extracellular traps such as eosinophils (EETs) and monocytes/macrophages (METs). (C) In the absence of immuno‐evasion, NETs can participate in larval killing by direct or indirect mechanisms not mutually exclusive: (i) Larvae are mechanically trapped by NETs. L3 are potentially exposed to a high concentration of “decorating” enzymes (NE, citrullinated histone H3 (H3Cit), MPO, and matrix metalloprotease 9 [MMP‐9]) or killed by other immune cells recruited to the traps and (ii) the cuticle of larvae is damaged by neutrophil enzymes such as NE, causing increased permeability to sytox green. (iii) NETs directly activate other immune cells, such as macrophage to potentiate their larvicidal activity. Immunofluorescence microscopy of Necator brasiliensis L3: (i) intravital imaging of larvae in the skin (CFSE stained, green) trapped by NETs stained with the DNA binding dye sytox blue (red). (ii) Larvae killed by NETs in vitro and stained with sytox green for 3 h. The damaged cuticle lets the otherwise impermeant dye through to stain the internal structures of the worm. Fluorescence intensity represented with the fire LUT in Fiji. (iii) Intravital imaging of neutrophils (Ly6G‐PE red) adhered to larvae (CFSE‐stained, green) surrounded by monocytes (Ly6C‐BV421, blue) 6 h post intradermal inoculation. For more details about the methodology, please see. The figure has been made using Biorender