Properties and function of Heligmosomoides polygurus secreted apyrases

Helminth parasite secreted molecules have been shown to modulate the host immune system to the extent of alleviating symptoms of immune disorders such as allergy and autoimmunity. Apyrases secreted by helminth parasites have potential immunoregulatory functions. They constitute a family of nucleotide-metabolising enzymes which can disturb purinergic signalling pathways of immune cells via hydrolysing inflammatory ATP released following tissue damage. In this thesis, the biochemical properties of the five apyrases secreted by the intestinal nematode Heligmosomoides polygyrus were elucidated via heterologous expression in the yeast Pichia pastoris. Results showed that the enzymes belonged to a group of calcium-dependent apyrases with a broad optimum pH and a broad substrate specificity, catalysing the hydrolysis of both nucleoside tri- and diphosphates. In an attempt to understand if any immune modulation was displayed by apyrases, in vivo studies were performed. Apyrase-1 and -3 were expressed in Trypanosoma musculi, a suitable in vivo vehicle for the expression of genes encoding secreted proteins of nematode parasites. Among the results shown, the transgenes grew faster compared to control trypanosomes, and splenocytes from mice infected with T. musculi expressing Apy-3 produced higher levels of IL-5 and IL-13. Both immunological and physiological factors appear to be responsible for these changes, suggesting that apyrases might modulate the immune response in addition to influencing the availability of extracellular purines for salvage by parasites. The effect of H. polygyrus secreted apyrases on type 2 immunity was also examined in this thesis during an acute model of allergic inflammation and during nematode infection. Intranasal administration of recombinant Apy-1 and Apy-3 did not seem to have an effect in regulating immunological responses, at least in the models tested. Further work is required to probe the precise function of apyrases secreted by parasitic nematodes and the possible immune modulatory effects exerted by these enzymes.Open Acces

Excretory-secretory products from adult helminth <i>Nippostrongylus brasiliensis</i> have <i>in vitro</i> bactericidal activity

Introduction. Intestinal helminths and microbiota share the same anatomical niche during infection and are likely to interact either directly or indirectly. Whether intestinal helminths employ bactericidal strategies that influence their microbial environment is not completely understood.Hypothesis. In the present study, the hypothesis that the adult hookworm Nippostrongylus brasiliensis produces molecules that impair bacterial growth in vitro, is tested.Aim. To investigate the in vitro bactericidal activity of Nippostrongylus brasiliensis against commensal and pathogenic bacteria.Methodology. The bactericidal effect of somatic extract and excretory-secretory products of adult Nippostrongylus brasiliensis on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae) bacteria was assessed using growth assays. Minimum inhibitory concentration and minimum bactericidal concentration assays were performed using excretory-secretory products released from the pathogen.Results. Broad-spectrum in vitro bactericidal activity in excretory-secretory products, but not somatic extract of adult Nippostrongylus brasiliensis was detected. The bactericidal activity of excretory-secretory products was concentration-dependent, maintained after heat treatment, and preserved after repeated freezing and thawing.Conclusion. The results of this study demonstrate that helminths such as Nippostrongylus brasiliensis release molecules via their excretory-secretory pathway that have broad-spectrum bactericidal activity. The mechanisms responsible for this bactericidal activity remain to be determined and further studies aimed at isolating and identifying active bactericidal molecules are needed

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function

Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor_biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions

Modulation of the immune response by nematode secreted acetylcholinesterase revealed by heterologous expression in Trypanosoma musculi

Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host

Expression of <i>N</i>. <i>brasiliensis</i> AChE B in <i>T</i>. <i>musculi</i>.

<p>(A) Detection by western blot. NbSP: Secreted products from <i>N</i>. <i>brasiliensis</i>; TmE: <i>T</i>. <i>musculi</i> extracts; TmSP: <i>T</i>. <i>musculi</i> secreted products. WT: Wild type trypanosomes; AChE; <i>T</i>. <i>musculi</i> expressing cytosolic AChE; sAChE: <i>T</i>. <i>musculi</i> expressing secreted AChE. (B) Tm-sAChE is glycosylated. Extracts and secreted products as in A), either with (+) or without (-) PNGase F treatment. Molecular mass markers are shown in kDa. (C) Tm-sAChE stained with antibody to <i>N</i>. <i>brasiliensis</i> AChE B and DAPI and viewed by indirect immunofluorescence. (D) Visualisation of AChE activity after non-denaturing gel electrophoresis, abbreviations as in panel A. (E) AChE activity measured by Ellman assay, abbreviations as in panel A. TmE: <i>T</i>. <i>musculi</i> extracts from 5 x 10⁵ trypanosomes; TmSP: <i>T</i>. <i>musculi</i> secreted products from 5 x 10⁴ trypanosomes cultured for 24 hrs. Data are shown as the mean ±SEM, assayed in triplicate.</p

Cellularity of spleens from mice infected with transgenic <i>T</i>. <i>musculi</i>.

<p>Phenotyping was performed by flow cytometry as described in Materials and Methods. Data are shown as the mean ± 1SEM (n = 5) and are representative of two independent experiments with 5 mice in each group. *p<0.05, **p<0.01. White bar = uninfected mice; Light grey bar = mice infected with Tm-luc; Dark grey bar = mice infected with Tm-sAChE.</p

Growth and survival of transgenic <i>T</i>. <i>musculi</i> in vitro and in vivo.

<p>(A) Growth <i>in vitro</i> of wild type <i>T</i>. <i>musculi</i> (WT), those engineered to express secreted AChE (Tm-sAChE) and expressing cytosolic luciferase (Tm-luc). (B) Survival of Tm-sAChE compared to Tm-luc <i>in vivo</i>. Parasitaemia in peripheral blood monitored over the course of infection of female BALB/c mice. Data are shown as the mean ±SEM (n = 5) and are representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. (C). Detection of AChE in serum of mice 8 days post-infection with Tm-luc and Tm-sAChE by activity-based gel assay [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005998#ppat.1005998.ref023" target="_blank">23</a>]. Samples of serum (5 μl) from individual mice were loaded in each lane.</p

Generation of transgenic <i>T</i>. <i>musculi</i> and expression of GFP.

<p>(A) Schematic depiction of expression vector pSSUGFP, utilising 5´ and 3´ regions to integrate into the 18S SSU rRNA gene locus, and both PFR and β-α tubulin intergenic regions to effect RNA processing of eGFP and blasticidin resistance genes. (B) Detection of eGFP expression by western blot. Lane 1: Wild type <i>T</i>. <i>musculi</i>; Lane 2: Parasites transformed with expression cassette in which eGFP was incorporated into the tubulin gene array (pTubGFP); Lane 3: Expression cassette with eGFP incorporated into the SSU rRNA locus (pSSUT7GFP) in a cell line with T7 RNA polymerase incorporated into the tubulin array (pT7polyNeo); Lane 4: Expression cassette with eGFP incorporated into the SSU rRNA locus alone (pSSUGFP). Molecular mass markers are shown in kDa. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005998#ppat.1005998.s002" target="_blank">S2 Fig</a> for different constructs. (C) Detection of eGFP expression by fluorescence microscopy: eGFP incorporated into the SSU rRNA locus.</p

Altered immune responses in mice infected with Tm-sAChE.

<p>(A) Specific antibody responses (end-point titres) to <i>T</i>. <i>musculi</i> at d14 post-infection. (B) Cytokine responses from splenocytes at d14 post-infection. Data are shown as the mean ±SEM (n = 5) and are representative of three independent experiments. **p<0.01, ***p<0.001. White bar = uninfected mice; Light grey bar = mice infected with Tm-luc; Dark grey bar = mice infected with Tm-sAChE.</p

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function.

From PubMed via Jisc Publications RouterHistory: received 2020-06-01, accepted 2021-03-19Publication status: ppublishFunder: Wellcome Trust; Grant(s): 204394Funder: British Heart Foundation; Grant(s): PG/12/36/29444Funder: Medical Research Council; Grant(s): MR/M003493/1, MR/R024812/1Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, and expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions. [Abstract copyright: Copyright © 2021 The Authors.