β-Glucan receptors on IL-4 activated macrophages are required for hookworm larvae recognition and trapping

Abstract

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for β-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed β-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize β-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.

Keywords: Nippostrongylus brasiliensis; glucan; hookworms; macrophages; recognition; trapping.

Figure 1

Competitive binding assays reveal the CLR ligands, mannan and laminarin, reduce Nb larval recognition by M(IL‐4). Bone marrow derived macrophages were generated from (a–d) C57BL/6 (B6) wild‐type, (e) mannose receptor deficient (MR^−/−), dectin‐2 deficient (Dec‐2^−/−), Fcγ receptor and complement 3 double deficient (FcgR/C3^−/−), or (f) dectin‐1 deficient (Dec‐1^−/−) mice. All macrophages were stimulated with IL‐4 (10 ng mL⁻¹) and pre‐incubated at 37°C for 1 h with various CLRs ligands at different concentrations as indicated (laminarin sourced from Sigma). Nb L3 were then added to the culture for 24 h. The percentage of larvae attacked by macrophages was then quantified by microscopy and manual counting of live and motile larvae. All experiments were performed 2 times independently, with n = 2 mice per experiment, and at least 3 technical replicates. (e) is pooled from 3 independent repeats. NS, not significant; untr, untreated; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 2

β‐Glucans, but not α‐glucans interfere with M(IL‐4) macrophage recognition of Nb L3 via non‐classical CLR pathways. (a‐d, f) Bone marrow derived macrophages were generated from C57BL/6 (B6) wild‐type mice. Macrophages were stimulated with IL‐4 (10 ng mL⁻¹) and (a–d) pre‐incubated at 37°C for 1 h with various CLRs ligands at different concentrations as indicated in the figure. (f) Nb L3 were incubated with laminarase (β‐(1→3)‐D‐glucanase), or 0.05 m Na acetate buffer control for 30 min, then extensively washed and (a–d, f) Nb L3 were then added to the culture for 24 h. The percentage of larvae attacked by macrophages was then quantified by microscopy and manual counting of live and motile larvae. Data are pooled from two independent experiments with technical triplicates. (e) Larvae were stained or not with aniline blue and imaged with a DAPI filter. Representative of 25 images, with three larvae per image. NS, not significant; untr, untreated; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 3

M(IL‐4) recognize larval β‐glucans on the Nb cuticle through expression of CD11b and EphA2 receptors. (a–e) Bone marrow derived macrophages were generated from C57BL/6 (B6) wild‐type, CD11b deficient (CD11b^−/−) or EphrinA2 deficient (EphA2^−/−) mice. Macrophages were stimulated with IL‐4 (10 ng mL⁻¹). (a–d) Nb L3 were then added to the culture for 24 h. The percentage of larvae attacked by macrophages was quantified 24 h later by microscopy and manual counting of live and motile larvae. To compare the effect of laminarin between macrophage genotypes, the percentage of attacked larvae by macrophages was normalized to unstimulated macrophages (no laminarin, UNS). Data are pooled from 2–4 independent experiments with technical triplicates. (e) Represents two independent experiments with technical quadruplicates and five total biological replicates. NS, not significant; untr, untreated; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.