Secreted filarial nematode galectins modulate host immune cells

Abstract

Lymphatic filariasis (LF) is a mosquito-borne disease caused by filarial nematodes including Brugia malayi. Over 860 million people worldwide are infected or at risk of infection in 72 endemic countries. The absence of a protective vaccine means that current control strategies rely on mass drug administration programs that utilize inadequate drugs that cannot effectively kill adult parasites, thus established infections are incurable. Progress to address deficiencies in the approach to LF control is hindered by a poor mechanistic understanding of host-parasite interactions, including mechanisms of host immunomodulation by the parasite, a critical adaptation for establishing and maintaining infections. The canonical type 2 host response to helminth infection characterized by anti-inflammatory and regulatory immune phenotypes is modified by filarial nematodes during chronic LF. Current efforts at identifying parasite-derived factors driving this modification focus on parasite excretory-secretory products (ESP), including extracellular vesicles (EVs). We have previously profiled the cargo of B. malayi EVs and identified B. malayi galectin-1 and galectin-2 as among the most abundant EV proteins. In this study we further investigated the function of these proteins. Sequence analysis of the parasite galectins revealed highest homology to mammalian galectin-9 and functional characterization identified similar substrate affinities consistent with this designation. Immunological assays showed that Bma-LEC-2 is a bioactive protein that can polarize macrophages to an alternatively activated phenotype and selectively induce apoptosis in Th1 cells. Our data shows that an abundantly secreted parasite galectin is immunomodulatory and induces phenotypes consistent with the modified type 2 response characteristic of chronic LF infection.

Keywords: brugia malayi; extracellular vesicles; filarial; galectin; immunomodulation.

Figure 1

Bma-LEC-1 and Bma-LEC-2 expression and secretion are sex- and life stage-specific. (A) Bma-LEC-1 and Bma-LEC-2 are most highly expressed in adult female stage parasites. (B) A monoclonal antibody raised against Bma-LEC-2 (34) can detect recombinant Bma-LEC-2 (approximately 32 kDa) at quantities as low as 3 ng. (C) Using this antibody, parasite galectin reactivity was predominantly detected in whole worm lysate of adult female parasites and to a lesser extent in adult males and microfilariae. (D) Parasites also secrete galectins into the extracellular milieu and galectin reactivity was detected in isolated extracellular vesicles (EVs) of adult female parasites and (E) in EV-depleted spent culture media of microfilariae and to a lesser extent adult female and male parasites. Representative images of three biologically distinct replicates.

Figure 2

Bma-LEC-1 and -2 are similar to other tandem-repeat type galectins. (A) Bma-LEC-1 and Bma-LEC-2 cluster with other tandem-repeat type galectins from diverse species. (B) Within the tandem-repeat galectins compared, Bma-LEC-1 and Bma-LEC-2 are most closely related to C. elegans LEC-1 and human/mouse galectin-9. Multiple sequence alignment of carbohydrate recognition domain 1 (C) and carbohydrate recognition domain 2 (D) characteristic of tandem-repeat type galectins revealed that Bma-LEC-1 had 83% identity to Celegans LEC-1 and 60% identity to human/mouse galectin-9, while Bma-LEC-2 had 71% identity to C. elegans LEC-1 and 59% and 58% identity to human/mouse galectin-9, respectively. Jalview (version 2.11.1.4) was used to create phylogenetic trees and multiple sequence alignment figures.

Figure 3

Expression of recombinant Bma-LEC-1 and Bma-LEC-2 in Sf21 cells. (A) DNA gel electrophoresis of Bma-LEC-1 and Bma-LEC-2 PCR amplicons from B. malayi adult female cDNA. (B) SDS-PAGE gel stained with Coomassie Blue showing elution of rBma-LEC-1 and rBma-LEC-2 expressing Sf21 cell lysates from initial nickel NTA resin purification and (C) after double purification using a FPLC system. Only clean FPLC fractions containing the protein of interest and no other debris or non-specific proteins were used for downstream assays. (D) Final confirmation of concentrated, double-purified rBma-LEC-1 and rBma-LEC-2 was conducted via an anti-6x His Tag western blot. Black arrowhead referencing tagged recombinant galectins of the expected 32 kDa size.

Figure 4

rBma-LEC-1/LEC-2 are functional homologs of mammalian galectin-9. (A) Hemagglutination inhibition (HI) assay was used to determine the carbohydrate binding specificities of rBma-LEC-1 and rBma-LEC-2. A panel of common carbohydrates was used to test if the recombinant proteins were functional galectins. A solid colored well indicates that the galectin present is not binding to the carbohydrate of interest but is binding the erythrocytes instead forming a lattice of erythrocytes on the bottom of the well. A well with a dot in the center indicates that the galectin present is binding the carbohydrate of interest not the erythrocytes allowing them to sediment at the bottom of the well. Both recombinant proteins were capable of binding 1 M galactose, 900 mM GlcNAc, 1 M lactose, and 32.6 mM LacNAc, each known galectin substrates. A similar sugar-binding profile was observed with human and mouse galectin-9. This representative image, from three distinct biological replicates, shows technical triplicates of each carbohydrate tested. (B) The binding sensitivity of galectins were analyzed by determining the minimal inhibitory concentration (MIC) of each carbohydrate solution. Two-fold serial dilutions of each carbohydrate were prepared in one row of a 96-well plate for each carbohydrate with the first well starting at the concentration indicated. Wells with a dot in the center indicate that the galectin is able to bind that concentration of the carbohydrate of interest. The MIC is determined at the first well where the galectin is no longer able to bind that concentration of carbohydrate as indicated by a solid colored well. rBma-LEC-1 had the highest affinity to all of the carbohydrate solutions tested and all galectins had the highest affinity to LacNAc. MIC are indicated at the end of the row. (C) A glycan binding array was used to compare rBma-LEC-1 and rBma-LEC-2 glycan binding profiles with those of human galectin -4, -8, -9 and -12 using a substrate of 584 glycan moieties. The glycan binding profiles of the parasite galectins are most similar to human galectin-8 and galectin-9. Glycan binding arrays are quantified by relative fluorescence units (RFU).An abbreviated version of this array data is presented with a full analysis found in Supplemental Materials 3. A corresponding key of glycan names associated with the numerical IDs is provided in Supplemental Materials 4.

Figure 5

Structures of rBma-LEC-1/LEC-2 highest affinity glycans. The binding affinities of rBma-LEC-1 and rBma-LEC-2 were investigated with a glycan binding array. rBma-LEC-1 had high affinity for alpha-galactose on a biantennary N-glycan, for blood group B on a biantennary N-glycan, and for blood group B on multiple O-glycan and N-glycan motifs. rBma-LEC-2 had high affinity for blood group B, for blood group B on biantennary N-glycans, for alpha-galactose on biantennary N-glycans, and for various O-glycan and N-glycan motifs. This table shows the structures of the top five highest binding glycans. 1: Gala1-3Galb1-4GlcNAcb1-2Mana1-6(Gala1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-GENR, 2: Gala1-3Galb1-4GlcNAcb1-2Mana1-6(Gala1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4GlcNAc-KVANKT, 3: Gala1-3(Fuca1-2)Galb1-4GlcNAcb1-2Mana1-6(Gala1-3(Fuca1-2)Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-NST, 4: Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-VANK, 5: Gala1-3(Fuca1-2)Galb1-4GlcNAc-CH2CH2NH2, 6: Gala1-3(Fuca1-2)Galb1-3GlcNAcb- CH2CH2NH2.

Figure 6

rBma-LEC-2 induces suppressive phenotypes in lymphoid and myeloid cells. (A) rBma-LEC-2, but not rBma-LEC-1, is a bioactive effector molecule that selectively induces apoptosis in Th1 cells, but not naïve T cells and (B) promotes polarization of macrophages to an alternatively activated phenotype as indicated by its increase in expression and (C) production of IL-10. These phenotypes are consistent with phenotypes induced by mammalian galectin-9. There is no synergistic effect in either inducing apoptosis of T cells or promoting polarization of macrophages when cells are treated with both human cytokines IL-4 and IL-13 and parasite-derived galectin. N = 3 (minimum) biologically distinct replicates. Mean ± SEM, *P < 0.05, **P < 0.01, ****P < 0.0001.