Exosomes secreted by a nematode parasite transfer small RNAs to mammalian cells and regulate genes of the innate immune system [Litomosoides sigmodontis]
ABSTRACT: In mammalian systems, extracellular small RNAs can operate in a paracrine manner to communicate information between cells, relying on transport within vesicles. “Foreign” small RNAs derived from bacteria, plants and parasites have also been detected in mammalian body fluids, sparking interest in whether these could mediate inter-species communication. However, there is no mechanistic framework for RNA-mediated interspecies communication and the active movement of RNA via vesicles has not been shown outside of mammals. Here we demonstrate that specific microRNAs and Y RNAs are packaged into vesicles secreted by a gastrointestinal nematode, Heligmosomoides polygyrus, which naturally infects mice. Total RNA was extracted from the serum of mice infected with Litomosoides sigmodontis at 60 days post infection
Project description:In mammalian systems, extracellular small RNAs can operate in a paracrine manner to communicate information between cells, relying on transport within vesicles. “Foreign” small RNAs derived from bacteria, plants and parasites have also been detected in mammalian body fluids, sparking interest in whether these could mediate inter-species communication. However, there is no mechanistic framework for RNA-mediated interspecies communication and the active movement of RNA via vesicles has not been shown outside of mammals. Here we demonstrate that specific microRNAs and Y RNAs are packaged into vesicles secreted by a gastrointestinal nematode, Heligmosomoides polygyrus, which naturally infects mice. Total RNA was extracted from the secretion product of adult worms and compared to the profile of small RNAs in adult worms, eggs and infective larvae.
Project description:We show that a gastrointestinal nematode that infects mice, Heligmosomoides polygyrus, secretes vesicles packaged with specific microRNAs (miRNAs) and Y RNAs as well as a nematode-specific Argonaute protein. The vesicles are of intestinal origin and are enriched for homologs of mammalian proteins found in exosomes, including heat shock proteins, tetraspanins and an ESCORT protein. The nematode-derived vesicles are internalized by mouse intestinal epithelial cells and mediate changes in expression of host genes involved in inflammation and immunity, including the receptor for the alarmin IL33 as well as a key regulator of MAPK signaling, DUSP1. A total of 12 samples were analyzed, from 4 different conditions: medium, exosome-treated, LPS and LPS+exosomes, each with 3 biological replicates. One of the 'medium' replicates was discarded due to outlier behaviour.
Project description:A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether T. cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16–40 nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells. Small RNAs profiles (16-40 nt) of epimastigote-derived extracellular vesicles, metacyclic trypomastigote-derived extracellular vesicles and metacyclic trypomastigote parental cells.
Project description:Mouse bone marrow derived macrophages were stimulated with L3 larvae of the helminth Heligmosomoides polygyrus (Hp) (500 L3 /1 mio cells) in the presence or absence of immune serum (1:50 v:v) from challenge-Hp-infected mice.
Project description:The intestinal helminth parasite Heligmosomoides polygyrus initiates infection in mice by penetrating the duodenal mucosa, where it develops while surrounded by a multicellular granulomatous infiltrate before emerging into the intestinal lumen. We examined early H. polygyrus infection to assess the epithelial response to disruption of the mucosal barrier. Unexpectedly, intestinal stem cell markers, including Lgr5 and Olfm4, were completely lost in crypts overlying larvae-associated granulomas. We sought to identify the mechanism by which the H. polygyrus granuloma represses the activity of intestinal stem cells. Overall design: CD45-negative EpCAM-positive CD44-positive crypt cells were sorted from duodenum of mice infected with H. polygyrus for six days. Crypt cells from granuloma punch biopsies was compared to crypt cells from non-granuloma punch biopsies.
Project description:Gene expression of Treg cells that have lost Foxp3 expression and acquired Il4 expression following adoptive transfer into T-cell deficient mice (HpTR-IL-4gfp+), cmpared to conventional Treg cells isolated from H. polygyrus-infected wild-type mice (HpTR) and Th2 cells generated from naïve T cells following adoptive transfer into H. polygyrus-infected T-cell deficient mice (nT-IL-4gfp+). Immunity to intestinal helminth infections requires the rapid activation of T helper 2 (Th2) cells. However, simultaneous expansion of regulatory CD4+Foxp3+ T (Treg) cells impedes protective responses, resulting in chronic infections. The ratio between regulatory and effector T cells can therefore determine the outcome of infection. The re-differentiation of Treg into T helper (Th) cells has been identified in hyper-inflammatory diseases. In this study, we asked whether ex-Treg Th2 cells develop and contribute to type 2 immunity. Using multi-gene reporter and fate-reporter systems we demonstrate that a significant proportion of Th2 cells derive from Foxp3+ cells following Heligmosomoides polygyrus infection and airway allergy. Ex-Foxp3 Th2 cells exhibit characteristic Th2 effector functions and provide immunity to H. polygyrus. Through selective deletion of Il4ra on Foxp3+ cells, we further demonstrate IL-4 is required for the development of ex-Foxp3 Th2 cells. Collectively, our findings indicate that converting Treg cells into Th2 cells could concomitantly enhance Th2 cells and limit Treg-mediated suppression. Overall design: Naïve T cells were FACS-sorted from naive wild-type mice. HpTR cells were FACS-sorted from H. polygyrus-infected mice. HpTR cells and nT cells were transferred to T-cell deficient mice infected with H. polygyrus. IL-4gfp-expressing cells were FACS-sorted from HpTR (HpTR-IL-4gfp+) or nT (nT-IL-4gfp+) recipients. 3 biological samples were obtained from 3 independent experiments for each sample group.