Secreted filarial nematode galectins modulate host immune cells

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.This article is published as Loghry HJ, Sondjaja NA, Minkler SJ and Kimber MJ (2022) Secreted filarial nematode galectins modulate host immune cells. Front. Immunol. 13:952104. DOI: 10.3389/fimmu.2022.952104. Copyright 2022 Loghry, Sondjaja, Minkler and Kimber. Attribution 4.0 International (CC BY 4.0). Posted with permission

Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum

Circular RNAs (circRNAs) are a recently identified RNA species with emerging functional roles as microRNA (miRNA) and protein sponges, regulators of gene transcription and translation, and modulators of fundamental biological processes including immunoregulation. Relevant to this study, circRNAs have recently been described in the parasitic nematode, Haemonchus contortus, suggesting they may have functionally important roles in parasites. Given their involvement in regulating biological processes, a better understanding of their role in parasites could be leveraged for future control efforts. Here, we report the use of next-generation sequencing to identify 1,997 distinct circRNAs expressed in adult female stages of the gastrointestinal parasitic nematode, Ascaris suum. We describe spatial expression in the ovary-enriched and body wall muscle, and also report circRNA presence in extracellular vesicles (EVs) secreted by the parasite into the external environment. Further, we used an in-silico approach to predict that a subset of Ascaris circRNAs bind both endogenous parasite miRNAs as well as human host miRNAs, suggesting they could be functional as both endogenous and exogenous miRNA sponges to alter gene expression. There was not a strong correlation between Ascaris circRNA length and endogenous miRNA interactions, indicating Ascaris circRNAs are enriched for Ascaris miRNA binding sites, but that human miRNAs were predicted form a more thermodynamically stable bond with Ascaris circRNAs. These results suggest that secreted circRNAs could be interacting with host miRNAs at the host-parasite interface and influencing host gene transcription. Lastly, although we have previously found that therapeutically relevant concentrations of the anthelmintic drug ivermectin inhibited EV release from parasitic nematodes, we did not observe a direct effect of ivermectin treatment on Ascaris circRNAs expression or secretion.This article is published as Minkler SJ, Loghry-Jansen HJ, Sondjaja NA and Kimber MJ (2022) Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum. Front. Genet. 13:884052. DOI: 10.3389/fgene.2022.884052. Copyright 2022 Minkler, Loghry-Jansen, Sondjaja and Kimber. Attribution 4.0 International (CC BY 4.0). Posted with permission

Towards nanovesicle-based disease diagnostics: a rapid single-step exosome assay within one hour through in situ immunomagnetic extraction and nanophotonic label-free detection

Exosomes have been considered as high-quality biomarkers for disease diagnosis, as they are secreted by cells into extracellular environments as nanovesicles with rich and unique molecular information, and can be isolated and enriched from clinical samples. However, most existing exosome assays, to date, require time-consuming isolation and purification procedures; the detection specificity and sensitivity are also in need of improvement for the realization of exosome-based disease diagnostics. This paper reports a unique exosome assay technology that enables completing both magnetic nanoparticle (MNP)-based exosome extraction and high-sensitivity photonic crystal (PC)-based label-free exosome detection in a single miniature vessel within one hour, while providing an improved sensitivity and selectivity. High specificity of the assay to membrane antigens is realized by functionalizing both the MNPs and the PC with specific antibodies. A low limit of detection on the order of 107 exosome particles per milliliter (volume) is achieved because the conjugated MNP–exosome nanocomplexes offer a larger index change on the PC surface, compared to the exosomes alone without using MNPs. Briefly, the single-step exosome assay involves (i) forming specific MNP–exosome nanocomplexes to enrich exosomes from complex samples directly on the PC surface at the bottom of the vessel, with a >500 enrichment factor, and (ii) subsequently, performing in situ quantification of the nanocomplexes using the PC biosensor. The present exosome assay method is validated in analyzing multiple membrane proteins of exosomes derived from murine macrophage cells with high selectivity and sensitivity, while requiring only about one hour. This assay technology will provide great potential for exosome-based disease diagnostics.This article is published as Zhang, Qinming, Hannah J. Loghry, Jingjing Qian, Michael J. Kimber, Liang Dong, and Meng Lu. "Towards nanovesicle-based disease diagnostics: a rapid single-step exosome assay within one hour through in situ immunomagnetic extraction and nanophotonic label-free detection." Lab on a Chip 21, no. 18 (2021): 3541-3549. DOI: 10.1039/D1LC00446H. Copyright 2021 The Royal Society of Chemistry. Attribution-NonCommercial 3.0 Unported (CC BY-NC 3.0). Posted with permission

Ivermectin inhibits extracellular vesicle secretion from parasitic nematodes

Lymphatic filariasis (LF) is a disease caused by parasitic filarial nematodes that is endemic in 49 countries of the world and affects or threatens over 890 million people. Strategies to control LF rely heavily on mass administration of anthelmintic drugs including ivermectin (IVM), a macrocyclic lactone drug considered an Essential Medicine by the WHO. However, despite its widespread use the therapeutic mode of action of IVM against filarial nematodes is not clear. We have previously reported that filarial nematodes secrete extracellular vesicles (EVs) and that their cargo has immunomodulatory properties. Here we investigate the effects of IVM and other anti-filarial drugs on parasitic nematode EV secretion, motility, and protein secretion. We show that inhibition of EV secretion was a specific property of IVM, which had consistent and significant inhibitory effects across nematode life stages and species, with the exception of male parasites. IVM inhibited EV secretion, but not parasite motility, at therapeutically relevant concentrations. Protein secretion was inhibited by IVM in the microfilariae stage, but not in any other stage tested. Our data provides evidence that inhibiting the secretion of immunomodulatory EVs by parasitic nematodes could explain, at least in part, IVM mode of action and provides a phenotype for novel drug discovery.This is the published version of the following article: Loghry, Hannah J., Wang Yuan, Mostafa Zamanian, Nicolas J. Wheeler, Timothy A. Day, and Michael J. Kimber. "Ivermectin inhibits extracellular vesicle secretion from parasitic nematodes." Journal of Extracellular Vesicles 10, no. 2 (2020): e12036. DOI: 10.1002/jev2.12036. Copyright 2020 The Authors. Attribution 4.0 International (CC BY 4.0). Posted with permission

Murine macrophages internalize parasite-derived EV by phagocytosis.

<p>Imaris 3D reconstructed confocal micrographs of murine J774A.1 macrophages. (A) Control macrophages showing internalization of PKH67-labeled EV (green) isolated from microfilaria (mf), L3, adult male (AM) and adult female (AF) worms in parallel with Fluoresbrite Carboxylate Microspheres (red, phagocytosis tracer). Macrophages are counterstained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple). (B) Macrophages treated with labeled EV (green) and microspheres (red) in the presence of 200 μM Dynasore. Absence of green and red indicates internalization of both EV and tracer are blocked. (C) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated transferrin (tracer, red) in the presence of 30 μM Chlorpromazine. Presence of green and absence of red indicates internalization of tracer is blocked but EV is not. (D) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated cholera toxin b (tracer, red) in the presence of 300 μM Genistein. Presence of green and general absence of red indicates internalization of tracer is generally blocked but EV is not. All Imaris images captured at magnification 68X, all scale bars 2 μm.</p

All <i>B</i>. <i>malayi</i> intra-mammalian life cycle stages release extracellular vesicles (EV).

<p>(A-D) EVs were isolated from spent culture media then sized and quantified using nanoparticle tracking analysis (NTA). NTA of EV preparations from three individual 24 hr cultures of microfilariae (A), L4 (B), adult male (C) and adult female parasites (D) are shown. Particle size is in nm. (E-F) Electron micrograph of representative adult male and female EV (white arrowheads), scale bar 100 nm.</p

Ivermectin inhibits EV release from filarial nematodes.

<p>(A) 1 μM IVM reduces EV release by <i>B</i>. <i>malayi</i> in vitro. Microfilaria (mf), L3, adult male (AM) and female (AF) were incubated in RPMI containing IVM or vehicle control. Media was collected after 24 hr and EV isolated and quantified. N = 3 (minimum), mean ± SEM, *P<0.05, **P<0.01, ****P<0.0001. (B) 1 μM IVM reduces EV release in vitro by IVM susceptible Missouri strain (MO) <i>D</i>. <i>immitis</i> L3 but not IVM reduced susceptibility JYD-34 strain. N = 3 (minimum), mean ± SEM, **P<0.01, ns not significant.</p

<i>Brugia</i> EV proteome contains markers of exosome biogenesis.

<p>(A-B) Gene Ontology (GO) analysis of adult male (A) and female (B) EV proteomes summarizing cellular location GO terms, including terms associated with the endosomal pathway. (C-D) GO analysis of adult male (C) and female (D) EV proteomes summarizing molecular function GO terms.</p

<i>B</i>. <i>malayi</i> microfilariae release EV from the excretory/secretory (ES) pore.

<p>Confocal scanning laser micrograph showing anti-Alix immunoreactivity (IR, green) focused at the ES pore (white arrowhead). Alix is frequently found in EV proteomes and considered an EV marker. Anti-Alix IR can be observed extending from the ES pore within a duct-like structure (inset). Worms were counterstained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple). Scale bar 20 μm.</p