Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes, and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently, we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis muscle-stage larvae (MSL) secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds to miRNA without exhibiting sequence specificity. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.

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.

Despite recent advances, animal-parasitic nematodes have thus far been largely refractory to genetic manipulation. We describe here a new approach providing proof of principle that CRISPR/Cas9-mediated gene editing of parasitic nematodes is achievable using vesicular stomatitis virus glycoprotein-pseudotyped extracellular vesicles for the delivery of Cas9-single guide ribonucleoprotein complexes. We demonstrate that extracellular vesicle-delivered ribonucleoproteins can be used to disrupt a secreted deoxyribonuclease in Nippostrogylus brasiliensis. Introduction of a repair template encoding multiple stop codons led to measurable reduction in expression of the targeted gene. Altered transcripts corresponding to the edited locus were detected by RT-PCR, demonstrating that vesicles can access cells of tissues actively expressing the gene of interest. These data provide evidence that this technique can be employed for targeted gene editing in N. brasiliensis, making this species genetically tractable for the first time, although further refinement will be necessary for routine and robust interrogation of gene function.

The Trichinella genus contains parasitic nematodes capable of infecting a wide range of hosts including mammals, birds and reptiles. Like other helminths, T. spiralis secretes a complex mixture of bioactive molecules capable of modulating its immediate surroundings and creating a hospitable environment for growth, survival and ultimately transmission. The constitution of these excretory-secretory products (ESPs) changes depending on the tissue niche and the specific stage of parasite development. Unique to T. spiralis is a true intracellular stage wherein larvae develop inside striated myotubes. Remarkably, the parasite larvae do not destroy the host cell but rather reprogram it to support their presence and growth. This transformation is largely mediated through stage-specific secretions released into the host cell cytoplasm. In this study, we apply state of the art proteomics and computational approaches to elucidate the composition and functions of muscle-stage T. spiralis ESPs. Moreover, we define a recurring, upstream motif associated with the stichosome, the main secretory organ of this worm, and can be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages.

Protease induced disruption of the epithelial barrier activates a type 2 innate immune lung response. Here we show that activation of the airway epithelium by papain or A. Alternata is regulated by lymphocyte-derived acetylcholine (Ach). Proteases were administered for three days by intranasal instillation in Ach Transferase (ChAT) reporter, Rag-gc KO and newly created RoRgtCreChATloxp mice. The cell recruitment and inflammatory lung response was analyzed 24 h later. Using ChAT reporter mice, we observe a rapid increase of ACh production by type 2 and 3 innate lymphoid cells (ILC) following papain challenge. To ascertain that the ACh+ILC3 subpopulation contributes to the response, we generated RoRgtCreChATloxp mice. We find decreased eosinophil recruitment, epithelial injury and airway hyperreactivity in ACh+ILC3 deficient mice upon acute papain or A. Alternata challenge. These findings identify that in addition of ILC2, ILC3-derived ACh as a significant contributor to papain induced allergic asthma.

Acetylcholine (ACh) from neuronal and non-neuronal sources plays an important role in the regulation of immune responses and is associated with the development of several disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell (ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection and therefore sought to determine whether this also plays a role in allergic inflammation. RoraCre+ChatLoxP mice (in which ILC2s cannot synthesize ACh) were exposed to an allergenic extract of the fungus Alternaria alternata, and immune responses in the airways and lung tissues were analyzed. Airway neutrophilia and expression of the neutrophil chemoattractants CXCL1 and CXCL2 were enhanced 24 h after exposure, suggesting that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic inflammation. The effect of non-selective depletion of ACh was examined by intranasal administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh in this manner resulted in a more profound enhancement of neutrophilia and chemokine expression, suggesting multiple cellular sources for the release of ACh. In contrast, depletion of ACh inhibited Alternaria-induced activation of ILC2s, suppressing the expression of IL-5, IL-13, and subsequent eosinophilia. Depletion of ACh reduced macrophages with an alternatively activated M2 phenotype and an increase in M1 macrophage marker expression. These data suggest that ACh regulates allergic airway inflammation in several ways, enhancing ILC2-driven eosinophilia but suppressing neutrophilia through reduced chemokine expression.

ABSTRACT CD4 T-cells require T-cell receptor (TCR) signalling for their activation and differentiation. Foxp3+ regulatory T-cells (Treg) are dependent on TCR signals for their differentiation and suppressive function. However, it is not fully known how TCR signalling controls the differentiation of polyclonal CD4 T-cells upon antigen recognition at the single-cell level in vivo. In this study, using Nr4a3-Tocky ( T imer- o f- c ell- k inetics-and-activit y ), which analyses temporal changes of antigen-reactive T-cells following TCR signalling, we investigated T-cell response to Spike protein fragments (S1a, S1b, S2a, and S2b) upon immunisation. We show that S1a and S2a induced the differentiation of PD1 hi CXCR5 + T follicular helper (Tfh) cells, which is related to CD4 T-cell immunogenicity. In contrast, S1b induced CD25 hi GITR hi PD-1 int Treg, which intermittently received TCR signalling. Using Foxp3-Tocky, which analyses Foxp3 transcriptional dynamics, the S1b-reactive Treg sustained Foxp3 transcription over time, which is a hallmark of activated Treg. Foxp3 fate-mapping showed that the S1b-reactive Treg were derived not from pre-existing thymic Treg, suggesting Foxp3 induction in non-Treg cells. Thus, the current study reveals temporally dynamic differentiation of CD4 T-cells and Treg upon immunisation in the polyclonal TCR repertoire.

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

Initiation of allergic airway pathology often depends on the protease activity of the inhaled allergen. Previous studies have shown that IL-17–driven neutrophil and eosinophil responses can promote allergic pathology1Kim J. Chang Y. Bae B. Sohn K.H. Cho S.H. Chung D.H. et al.Innate immune crosstalk in asthmatic airways: innate lymphoid cells coordinate polarization of lung macrophages.J Allergy Clin Immunol. 2019; 143: 1769-1782.e11Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar and are associated with an initial epithelial release of IL-23, which is accepted as important in promoting TH17 and group 3 innate lymphoid cell (ILC3) responses. ILC3s are a developmentally and phenotypically diverse innate lymphoid cell (ILC) subset that includes natural killer (NK) cell receptor (NCR)-positive and NCR-negative populations (NCR+ ILC3s and NCR– ILC3s) as well as CCR6+ lymphoid tissue inducer cells. However, all ILC3s are defined by expression of transcript variant 2 of RORC, encoding retinoid-related orphan receptor γt (RoRγt) and production of IL-17 and IL-22.1Kim J. Chang Y. Bae B. Sohn K.H. Cho S.H. Chung D.H. et al.Innate immune crosstalk in asthmatic airways: innate lymphoid cells coordinate polarization of lung macrophages.J Allergy Clin Immunol. 2019; 143: 1769-1782.e11Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,2Ardain A. Domingo-Gonzalez R. Das S. Kazer S.W. Howard N.C. Singh A. et al.Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.Nature. 2019; 570: 528-532Crossref PubMed Scopus (95) Google Scholar Work carried out in murine models has resulted in ILC3s emerging as critical regulators of infectious2Ardain A. Domingo-Gonzalez R. Das S. Kazer S.W. Howard N.C. Singh A. et al.Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.Nature. 2019; 570: 528-532Crossref PubMed Scopus (95) Google Scholar and noninfectious1Kim J. Chang Y. Bae B. Sohn K.H. Cho S.H. Chung D.H. et al.Innate immune crosstalk in asthmatic airways: innate lymphoid cells coordinate polarization of lung macrophages.J Allergy Clin Immunol. 2019; 143: 1769-1782.e11Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar pulmonary diseases despite ILC3s representing only a minor lung immune cell population in mice. In contrast, ILC3s are the major ILC population in the human lung.3De Grove K.C. Provoost S. Verhamme F.M. Bracke K.R. Joos G.F. Maes T. et al.Characterization and quantification of innate lymphoid cell subsets in human lung.PLoS One. 2016; 11e0145961Crossref PubMed Scopus (96) Google Scholar Importantly, ILC3-associated preclinical phenotypes are reflective of observations in humans.2Ardain A. Domingo-Gonzalez R. Das S. Kazer S.W. Howard N.C. Singh A. et al.Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.Nature. 2019; 570: 528-532Crossref PubMed Scopus (95) Google Scholar,4Jonckheere A.C. Bullens D.M.A. Seys S.F. Innate lymphoid cells in asthma: pathophysiological insights from murine models to human asthma phenotypes.Curr Opin Allergy Clin Immunol. 2019; 19: 53-60Crossref PubMed Scopus (21) Google Scholar However, our understanding of the molecular machinery enabling ILC3s to exact their influence on lung immunity is incomplete. In this study, we have demonstrated that expansion of lung ILC3s occurs in response to the protease papain and that these cells promote an IL-17–associated lung pathology. Critically, we have shown that induction of papain-driven pathology is strongly associated with ILC3 synthesis of acetylcholine (ACh). We have previously identified ACh responsiveness by immune cells in the lung as being important for CD4+ T-cell–driven adaptive immunity to Nippostrongylus brasiliensis infection.5Darby M. Schnoeller C. Vira A. Culley F.J. Bobat S. Logan E. et al.The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection.PLoS Pathog. 2015; 11e1004636Crossref PubMed Scopus (30) Google Scholar Here, we have extended this insight by demonstrating that ACh from lineage-negative (Lin–) CD127+ lymphocytes expressing RoRγt is instrumental for promoting protease induction of allergic inflammation. This identifies a new paradigm for how ILC3s and ACh contribute to early promotion of allergic pathology that is distinct from our traditional understanding of ACh-driven neuromuscular interactions causing allergic pulmonary airway resistance. We have identified association of ILC3s with protease-induced lung pathology following acute papain challenge of wild-type (WT) C57BL/6 mice. Papain challenge increased lung concentrations of IL-13, IL-17A, IL-22, and IL-23 in comparison with the concentrations in saline-challenged mice (Fig 1, A). IL-17– and IL-23–promoted pathology suggests a RoRγt-driven inflammation. Papain challenge of RoRγt–green fluorescent protein (GFP) reporter mice demonstrated a significant expansion of the numbers of ILC3s (Lin–CD45+CD127+ICOS–RoRγt-GFP+), group 2 ILCs (ILC2s) (Lin–CD45+CD127+ICOS+RoRγt-GFP–), and CD3+CD4+ RoRγt-GFP+ T cells relative to the numbers in saline-treated controls (Fig 1, B and see Fig E1 in this article's Online Repository at www.jacionline.org). Restimulation and intracellular cytokine capture of lung CD4+ T cells from papain-challenged mice detected increased levels of IL-5 and IL-13 but not IL-17 when compared with the levels in saline-treated controls (Fig 1, C). However, in CD45+CD3–Lin– cells, in addition to increased levels of IL-5 and IL-13, a raised IL-17 level was detected in papain-challenged mice when compared with the levels in saline-treated controls (Fig 1, C). Moreover, anti-CD3 depletion of T cells did not protect against and in fact promoted papain-driven pathology (see Fig E2 in this article's Online Repository at www.jacionline.org). These findings support IL-17–driven pathology as being independent of RoRγt+ TH17 T-cell IL-17 production. To test a requirement for any T-cell (and B-cell) contribution to IL-23/IL-17–promoted pathology, we challenged RAG2–/– mice with papain in the presence or absence of an IL-23–neutralizing mAb (anti–IL-23) (Fig 1, D). Decreased lung inflammation in IL-23–depleted RAG2–/– mice was revealed by histologic analysis as well as by reduced detection of Evans blue (EB) leakage into bronchoalveolar lavage fluid (BALF) (Fig 1, D) when compared with that in isotype-treated RAG2–/– mice. BALF immune cell infiltration was reduced in all immune cell populations, and tissue levels of IL-17A (but not IL-13) were lower in the IL-23–depleted RAG2–/– mice (Fig 1, D). This abrogation of papain-induced allergic inflammation in anti–IL-23–treated RAG2–/– mice supports ILC3s as being a key contributing lymphoid cell population driving protease-mediated lung inflammation. To further characterize the input of ILC3s to allergic airway pathology, we compared pulmonary responses to papain in WT and Rorc–/– mice (Fig 1, E). Rorc–/– mice did not show significant baseline differences from C57Bl/6 mice in terms of BALF cell composition (see Fig E3 in this article's Online Repository at www.jacionline.org), but histologic analysis of lung sections revealed decreased inflammation and detection of EB leakage into the BALF in Rorc–/– mice challenged with papain versus in WT mice (Fig 1, E). Total BALF immune cell infiltration was also reduced for all immune cell populations (Fig 1, E). Moreover, detection of both IL-13 and IL-17A, as well as IL-22, was reduced in Rorc–/– mice (Fig 1, E). In agreement with findings by others6Lim A.I. Li Y. Lopez-Lastra S. Stadhouders R. Paul F. Casrouge A. et al.Systemic human ILC precursors provide a substrate for tissue ILC differentiation.Cell. 2017; 168: 1086-1100.e10Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar Rorc–/– mice had expanded numbers of ILC2s (Lin–CD45+CD127+ICOS+) when compared with the numbers in WT mice (Fig 1, E). However, as expected, detection of ILC3 subsets such as NCR+ ILC3s (Lin–CD45+CD127+ICOS–NKp46+) in Rorc–/– mice was acutely reduced as opposed to in WT mice; the small number of cells detected were most likely to be non-NK, non–RoRγt-expressing group 1 ILCs (ILC1s). This body of work identifies a previously unappreciated, T-cell–independent role for IL-23–responsive ILC3s in contributing to the onset of papain-driven lung pathology. An additional striking feature of these results was protection from cholinergic-promoted airway resistance during papain challenge in the absence of Rorc expression (Fig 1, E). Lymphocytes are important responders to, and sources of, neurotransmitters. For example, the ILC2 response to the neurotransmitter neuromedin U is critical for inducing type 2 immunity,7Cardoso V. Chesne J. Ribeiro H. Garcia-Cassani B. Carvalho T. Bouchery T. et al.Neuronal regulation of type 2 innate lymphoid cells via neuromedin U.Nature. 2017; 549: 277-281Crossref PubMed Scopus (305) Google Scholar and production of ACh by immune cells following type 2 immune challenge can promote host type 2 immune responses.8Roberts L.B. The influence of non-neuronal cholinergic signalling on the type 2 immune response. Imperial College London, London, UK2017Google Scholar Moreover, ACh-producing T cells can contribute to control of chronic viral infection,9Cox M.A. Duncan G.S. Lin G.H.Y. Steinberg B.E. Yu L.X. Brenner D. et al.Choline acetyltransferase-expressing T cells are required to control chronic viral infection.Science. 2019; 363: 639-644Crossref PubMed Scopus (45) Google Scholar and CD4+ T-cell responses to ACh via the M3 muscarinic receptor are required for optimal adaptive immunity to helminth and bacterial infections.5Darby M. Schnoeller C. Vira A. Culley F.J. Bobat S. Logan E. et al.The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection.PLoS Pathog. 2015; 11e1004636Crossref PubMed Scopus (30) Google Scholar In the spleen, lymphocytes are major effectors of the cholinergic anti-inflammatory pathway through their synthesis and release of ACh, which downregulates inflammation. ILC3 responses to cholinergic stimulation can also contribute to the cholinergic anti-inflammatory pathway by regulating neutrophilia in sepsis models. However, whether ILC3s themselves may be an immune cell–derived source of ACh capable of regulating immunity has not previously been investigated. To identify whether lung ILC3s may produce ACh during acute protease-induced inflammation, we challenged ChAT(BAC)-eGFP reporter mice with papain. In addition to increased choline acetyltransferase (ChAT) production by CD3+CD4+ cells (and a trend toward increased production by Lin–CD45+CD127+ICOS+ ILC2s), we identified an increase in the ChAT-expressing ILC3-enriched (Lin–CD45+CD127+ICOS–) population, but with no effect on the number of NK (CD3–DX5+) cells in the lungs of papain-challenged mice (Fig 2, A). Therefore, RoRγt-expressing ILC3s increase synthesis of ACh following papain challenge. To test whether ILC3 synthesis of ACh contributes to papain-driven pathology, we generated RoRγtCreChATloxp mice. These mice lack the ability to generate ACh following deletion of ChAT in RoRγt-expressing cells. Papain challenge of RoRγtCreChATloxp mice resulted in decreased histologic detection of inflammation and vascular leakage of EB in BALF compared with in the BALF of ChATloxp mice (Fig 2, B), along with reduced methacholine-induced airway resistance and reduced numbers of neutrophils and eosinophils in the BALF (Fig 2, B). Detection of cytokines in lung homogenates identified reduced IL-13 and IL-17A levels as well as reduced IL-22 levels, in RoRγtCreChATloxp mice as compared with in the controls (Fig 2, B). Therefore, disruption of ChAT in RoRγt+ cells was sufficient to recapitulate the reduced pathology seen in papain-challenged Rorc–/– mice (Fig 1). Quantification of ILCs revealed equivalent ILC2 (Lin–CD45+CD127+ICOS+) numbers but decreased NCR+ ILC3 (Lin–CD45+CD127+ICOS–NKp46+) numbers between papain-challenged WT and RoRγtCreChATloxp mice (Fig 2, B). An equivalent phenotype was demonstrated following Alternaria alternata extract–driven acute allergic inflammation (Fig 2, C). As with papain, A alternata extract challenge of RoRγtCreChATloxp mice resulted in decreased histologic detection of inflammation and vascular leakage of EB in BALF along with reduced methacholine-induced airway resistance, reduced numbers of neutrophils and eosinophils in the BALF, and reduced detection of IL-13 and IL-17A in lung homogenates compared with in ChATloxp mice (Fig 2, C). In summary, following acute protease challenge, we found raised IL-13, IL-23, IL-22, and IL-17A expression in the lung and increased numbers of ILC3s. Papain challenge did not induce elevated CD4+ T-cell IL-17 levels irrespective of raised RoRγt+CD4+ T-cell numbers. Moreover, in the absence of T cells, papain-induced lung inflammation was maintained but abrogated when IL-23 or RoRγt function was disrupted. This strongly supports ILC3 expansion in the lung as a driving factor in IL-17–associated inflammation following an acute protease lung challenge. Reduction in airway cholinergic responsiveness led us to investigate whether ILC3s were a physiologically relevant source of ACh following papain challenge. We tested this by generating RoRγtCreChATloxp mice that lack the ability to generate ACh in RoRγt-expressing cells. Remarkably, this ILC3-biased disruption of ChAT expression protected against pathology to an extent equivalent to that seen in Rorc–/– mice. This identifies RoRγt+ cell expression of ChAT as an important component in the promotion of protease-mediated allergic lung pathology. Indeed, these data support ILC3s expressing ACh as playing a central role in initiation of the IL-17–promoted allergic inflammatory cascade. These findings place ILC3 synthesis of ACh as a central requirement for allergic lung inflammation, adding a critical new paradigm to our understanding of cholinergic responses in driving allergic lung inflammation and pathology. We would like to acknowledge Elodie Culerier for her technical assistance and Marc Le Bert (INEM UMR7355 Experimental and Molecular Immunology and Neurogenetics, CNRS and University of Orleans) for expert advice on mouse genetics. All mice used in this study were 6- to 12-week-old C57BL/6 background mice. The mouse strains used were C57BL/6, RAG2–/–, ChAT(BAC)-eGFP,E1Tallini Y.N. Shui B. Greene K.S. Deng K.Y. Doran R. Fisher P.J. et al.BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.Physiol Genomics. 2006; 27: 391-397Crossref PubMed Scopus (129) Google Scholar RoRγt-eGFP,E2Eberl G. Marmon S. Sunshine M.J. Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells.Nat Immunol. 2004; 5: 64-73Crossref PubMed Scopus (778) Google Scholar RoRγt-KO (Rorc–/–),E3Sun Z. Unutmaz D. Zou Y.R. Sunshine M.J. Pierani A. Brenner-Morton S. et al.Requirement for RORgamma in thymocyte survival and lymphoid organ development.Science. 2000; 288: 2369-2373Crossref PubMed Scopus (588) Google Scholar, E4Eberl G. Littman D.R. Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells.Science. 2004; 305: 248-251Crossref PubMed Scopus (402) Google Scholar RoRγtCre,E3Sun Z. Unutmaz D. Zou Y.R. Sunshine M.J. Pierani A. Brenner-Morton S. et al.Requirement for RORgamma in thymocyte survival and lymphoid organ development.Science. 2000; 288: 2369-2373Crossref PubMed Scopus (588) Google Scholar and ChATloxp.E5Misgeld T. Burgess R.W. Lewis R.M. Cunningham J.M. Lichtman J.W. Sanes J.R. Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase.Neuron. 2002; 36: 635-648Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar Rorc–/– mice are deficient in expression of transcript variant 2 of RORC, encoding RoRγt. RoRγt is necessary for the development of lymph nodes and Peyer patches, which fail to develop in Rorc–/– mice.E4Eberl G. Littman D.R. Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells.Science. 2004; 305: 248-251Crossref PubMed Scopus (402) Google Scholar RoRγt is expressed by many cells, including immature double-positive (CD4+CD8+) αβ thymocytes, RoRγt+ TH17 T cells, and ILC3s, which comprise NCR+ ILC3s and NCR– ILC3s, as well as CCR6+ lymphoid tissue inducer cells. RoRγtCre and ChATloxp mice were crossed over 5 generations to generate RoRγtCreChATfl mice. The ChAT(BAC)-eGFP mouse has been demonstrated to faithfully report ChAT expression and ACh synthesis capacity across a number of hematopoietic immune cell types.E1Tallini Y.N. Shui B. Greene K.S. Deng K.Y. Doran R. Fisher P.J. et al.BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.Physiol Genomics. 2006; 27: 391-397Crossref PubMed Scopus (129) Google Scholar As the major rate-limiting enzyme of crucial significance for ACh synthesis, demonstration of ChAT expression by a specific cell type is widely considered to reflect the cholinergic-synthesizing nature of the cell. The mice were anesthetized by isoflurane followed by intranasal administration of 25 μg of papain (Calbiochem, Darmstadt, Germany) in 40 μL of saline solution per mouse once per day on days 1, 2, and 3. On day 4 mice, were humanely killed by CO2 inhalation 24 hours after the final administration of papain. A alternata extract was administered daily for 3 days at a dose of 25 μg per mouse by the endotracheal route under light isoflurane anaesthesia, before the mice were humanely killed on day 4 by CO2 inhalation. Anti–IL-23 antibody or isotype control (Bio X cell, Lebanon, NH; BR0313 or rat IgG1) was given daily 1 hour before papain challenge at a dose of 200 μg per mouse via the endotracheal route. Anti-CD3 (Biolegend, San Diego, Calif; catalog no. BL100208) antibody or isotype controls was given via intraperitoneal injection daily 1 hour before papain challenge at a rate of 50 μg per mouse. After the mice were humanely killed, BALF was collected before cardiac perfusion with ISOTON II (acid-free balanced electrolyte solution, Beckman Coulter, Krefeld, Germany), after which the lungs were collected and sampled for analyses. All of the animal experimental protocols complied with French ethical and animal experiments regulations (see Charte Nationale, Code Rural R 214-122, 214-124 and European Union Directive 86/609/EEC) and were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans, registered (No. 3) by the French National Committee of Ethical Reflexion for Animal Experimentation (CLE CNRS Campus Orleans 2013-1006). All of the South African experiments were carried out in accordance with South African Veterinary Council regulations and were approved by the University of Cape Town Faculty of Health Sciences Animal Ethics Committee. The left lobe of lung was fixed in 4% buffered formaldehyde and paraffin embedded under standard conditions. Tissue sections (3-μm) were stained with standard hematoxylin and eosin and periodic acid–Schiff. The histologic score of the pathology was determined by a semiquantitative assessment on a scale of 0 to 5 for cell infiltration (with increasing extent). The slides were blindly examined by 2 investigators with a Leica microscope (Leica, Solms, Germany). Bronchoalveolar lavage was performed by 4 lavages of lung with 500 μL of saline solution via a cannula introduced into the mouse trachea. BALF samples were centrifuged at 400 g for 10 minutes at 4°C, the supernatants were stored at –20°C for analysis, and pellets were recovered to prepare Cytospin (Thermo Scientific, Waltham, Mass) on glass slides followed by staining with Diff-Quik stain (Merz & Dade AG, Dudingen, Switzerland). Differential cell counts were performed with at least 300 cells. Vascular leakage was quantified by protein and EB concentration in the BALF. EB in BALF was measured 45 minutes after intravenous injection of 0.3% EB; the measurement was performed by absorbance at 460 nm, as described elsewhere.E6Michaudel C. Mackowiak C. Maillet I. Fauconnier L. Akdis C.A. Sokolowska M. et al.Ozone exposure induces respiratory barrier biphasic injury and inflammation controlled by IL-33.J Allergy Clin Immunol. 2018; 142: 942-958Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Extravasation was expressed as micrograms of EB per milliliter (μg of EB/mL) of BALF supernatant. Lungs were digested in RPMI 1640 medium containing 100 U/mL of penicillin, 100 U/mL of streptomycin, 1 mg/mL of DNase I (Sigma, St Louis, Mo), and 125 μg of liberase (Roche, Basel, Switzerland) for 1 hour at 37°C under rotation. After digestion, RPMI 1640 medium supplemented with 10% FCS was added. Cells were dissociated by passage through a 70-μm cell strainer and centrifuged at 400 g for 5 minutes at 4°C. Pellets were resuspended in red blood cell lysis buffer (Stem Cell Technologies, Vancouver, British Columbia, Canada) and incubated for 10 minutes on ice. Lysis was stopped by addition of RPMI 1640 medium and centrifuged again at 400 g for 5 minutes at 4°C. Pellets were resuspended in RPMI 1640 medium supplemented with 10% FCS and passed through a 40-μm cell strainer. ILC2s were identified by using a Lin cocktail, CD45, CD127, and ICOS panel. ILC3s were identified by using a panel containing a Lin cocktail, CD45, CD127, ICOS, and NKp46 or RoRγt, as indicated. Cytokine-expressing T cells were identified by using a panel containing CD3, CD4, IL-5, IL-13, or IL-17, as indicated. NK cells were identified by using a panel containing CD3 and DX5. All FACS antibodies were from Biolegend. Homogenized lungs were tested for IL-13 and IL-17 by using commercial ELISA kits (eBiosciences, San Diego, Calif) according to the manufacturer's instructions. For invasive measurement of dynamic resistance, mice were anesthetized by intraperitoneal injection of solution containing ketamine (100 mg/kg, Merial, Duluth, Ga) and xylazine (10 mg/kg, Bayer, Leverkusen, Germany), paralyzed by using D-tubocuranine (0.125%, Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per minute with a tidal volume of 0.2 mL and a positive end-expiratory pressure of 2 mL of H2O. Increasing concentrations of aerosolized methacholine (9.375, 18.75, 37.5, 75, and 150 mg/mL) were administered. Resistance was recorded by using an invasive plethysmograph (Buxco, London, United Kingdom). Baseline resistance was restored before administration of the subsequent doses of methacholine.E7Madouri F. Chenuet P. Beuraud C. Fauconnier L. Marchiol T. Rouxel N. et al.Protein kinase Ctheta controls type 2 innate lymphoid cell and TH2 responses to house dust mite allergen.J Allergy Clin Immunol. 2017; 139: 1650-1666Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Data were analyzed by using Prism software, version 5 or 6 (GraphPad Software, San Diego, Calif). Either a Mann-Whitney t test or the parametric 1-way ANOVA test with Bonferroni multiple-comparison was used to assess significance. Values are expressed as means ± SDs.Fig E2CD3-depleted ChAT-GFP mice do not show reduced inflammation to papain. Mice were treated daily with anti-CD3 antibody administered intraperitoneally 1 hour before papain challenge at a dose of 50 μg/mouse. BALF cellular infiltration was assessed 24 hours after final administration of papain. BAL, Bronchoalveolar lavage fluid; Eos., eosinophil; Gra., granulocyte; Lymp, lymphocyte; Mono., monocyte.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Rorc–/– mice do not show significant baseline differences from C57Bl/6 mice in terms of BALF cell composition. BALF immune cell infiltration and levels of lung ILC2s (Lin–CD45+CD127+ICOS+) and NCR+ ILC3s (Lin–CD45+CD127+ICOS–NKp46+) in saline-treated C57Bl/6 and Rorc–/– mice were analyzed by flow cytometry. BAL, Bronchoalveolar lavage fluid; Eos., eosinophil; Gra., granulocyte; Lymp, lymphocyte; Mono., monocyte.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.

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.

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.

Larvae of the cestodes Taenia solium and Taenia crassiceps infect the central nervous system of humans. Taenia solium larvae in the brain cause neurocysticercosis, the leading cause of adult-acquired epilepsy worldwide. Relatively little is understood about how cestode-derived products modulate host neural and immune signalling. Acetylcholinesterases, a class of enzyme that breaks down acetylcholine, are produced by a host of parasitic worms to aid their survival in the host. Acetylcholine is an important signalling molecule in both the human nervous and immune systems, with powerful modulatory effects on the excitability of cortical networks. Therefore, it is important to establish whether cestode derived acetylcholinesterases may alter host neuronal cholinergic signalling. Here we make use of multiple techniques to profile acetylcholinesterase activity in different extracts of both Taenia crassiceps and Taenia solium larvae. We find that the larvae of both species contain substantial acetylcholinesterase activity. However, acetylcholinesterase activity is lower in Taenia solium as compared to Taenia crassiceps larvae. Further, whilst we observed acetylcholinesterase activity in all fractions of Taenia crassiceps larvae, including on the membrane surface and in the excreted/secreted extracts, we could not identify acetylcholinesterases on the membrane surface or in the excreted/secreted extracts of Taenia solium larvae. Bioinformatic analysis revealed conservation of the functional protein domains in the Taenia solium acetylcholinesterases, when compared to the homologous human sequence. Finally, using whole-cell patch clamp recordings in rat hippocampal brain slice cultures, we demonstrate that Taenia larval derived acetylcholinesterases can break down acetylcholine at a concentration which induces changes in neuronal signalling. Together, these findings highlight the possibility that Taenia larval acetylcholinesterases can interfere with cholinergic signalling in the host, potentially contributing to pathogenesis in neurocysticercosis.

Trichinella spiralismuscle stage larvae (mL1) produce excretory-secreted products (ESPs), a complex mixture of protein, which are believed to be important for establishing or maintaining an infection niche within skeletal muscle and the intestine. Studies of both whole ESPs and individual cloned proteins have shown that some ESPs are potent immunogens capable of eliciting protective immune responses. Here we describe two novel proteins,Secreted fromMuscle stageLarvae SML-4 and SML-5 which are 15 kDa and 12 kDa respectively. The genes encoding these proteins are highly conserved within the Trichinellids, are constituents of mL1 ESP and localized in the parasite stichosome. While SML-5 is only expressed in mL1 and early stages of adult nematode development, SML-4 is a tyvosylated glycoprotein also produced by adult nematodes, indicating it may have a function in the enteral phase of the infection. Vaccination with these proteins resulted in an impaired establishment of adult stages and consequently a reduction in the burden of mL1 in BALB/c mice. This suggests that both proteins may be important for establishment of parasite infection of the intestine and are prophylactic vaccine candidates.

Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment, largely encapsulated within small vesicles. Parasite-secreted material often contains microRNAs (miRNAs), raising the possibility that they might regulate host genes in target cells. Here we characterise secreted RNAs from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly, T. spiralis muscle stage larvae, which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests that RNAs may be secreted without encapsulation in vesicles, with implications for the biology of T. spiralis infection.

Infection with parasitic helminths can imprint the immune system to modulate bystander inflammatory processes. Bystander or virtual memory CD8+ T cells (TVM) are non-conventional T cells displaying memory properties that can be generated through responsiveness to interleukin (IL)-4. However, it is not clear if helminth-induced type 2 immunity functionally affects the TVM compartment. Here, we show that helminths expand CD44hiCD62LhiCXCR3hiCD49dlo TVM cells through direct IL-4 signaling in CD8+ T cells. Importantly, helminth-mediated conditioning of TVM cells provided enhanced control of acute respiratory infection with the murid gammaherpesvirus 4 (MuHV-4). This enhanced control of MuHV-4 infection could further be explained by an increase in antigen-specific CD8+ T cell effector responses in the lung and was directly dependent on IL-4 signaling. These results demonstrate that IL-4 during helminth infection can non-specifically condition CD8+ T cells, leading to a subsequently raised antigen-specific CD8+ T cell activation that enhances control of viral infection.

Methylation at the 5 position of cytosine in DNA (5meC) is a key epigenetic mark in eukaryotes. Once introduced, 5meC can be maintained through DNA replication by the activity of 'maintenance' DNA methyltransferases (DNMTs). Despite their ancient origin, DNA methylation pathways differ widely across animals, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. We used comparative epigenomics to investigate the evolution of DNA methylation. Although the model nematode Caenorhabditis elegans lacks DNA methylation, more basal nematodes retain cytosine DNA methylation, which is targeted to repeat loci. We found that DNA methylation coevolved with the DNA alkylation repair enzyme ALKB2 across eukaryotes. In addition, we found that DNMTs introduced the toxic lesion 3-methylcytosine into DNA both in vitro and in vivo. Alkylation damage is therefore intrinsically associated with DNMT activity, and this may promote the loss of DNA methylation in many species.