TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus

Funding: This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001220), the UK Medical Research Council (FC001220), and the Wellcome Trust (FC001200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments We are indebted to The Francis Crick Institute Flow Cytometry facility, and in particular Bhavik Patel, Graham Preece, Wayne Turnbull and Phil Hobson. We would also like to thank The Francis Crick Institute Procedural Service Section for production of GA lines and Biological Services, especially Trisha Norton, Keith Williams and Adebambo Adekoya for animal husbandry and technical support; to Riccardo Guidi for constructive discussions and technical assistance. We would like to thank Gitta Stockinger and AhR Immunity Laboratory for providing technical support and reagents throughout this study. We also thank Richard Rance and the Wellcome Trust Sanger Institute’s 454 pyrosequencing team for generating 16S rRNA gene data.Peer reviewedPublisher PD

Single cell analyses identify a highly regenerative and homogenous human CD34+ hematopoietic stem cell population

The heterogeneous nature of human CD34+ hematopoietic stem cells (HSCs) has hampered our understanding of the cellular and molecular trajectories that HSCs navigate during lineage commitment. Using various platforms including single cell RNA-sequencing and extensive xenotransplantation, we have uncovered an uncharacterized human CD34+ HSC population. These CD34+EPCR+(CD38/CD45RA)− (simply as EPCR+) HSCs have a high repopulating and self-renewal abilities, reaching a stem cell frequency of ~1 in 3 cells, the highest described to date. Their unique transcriptomic wiring in which many gene modules associated with differentiated cell lineages confers their multilineage lineage output both in vivo and in vitro. At the single cell level, EPCR+ HSCs are the most transcriptomically and functionally homogenous human HSC population defined to date and can also be easily identified in post-natal tissues. Therefore, this EPCR+ population not only offers a high human HSC resolution but also a well-structured human hematopoietic hierarchical organization at the most primitive level

Epithelial-Cell-Derived Phospholipase A2 Group 1B Is an Endogenous Anthelmintic.

Immunity to intestinal helminth infections has been well studied, but the mechanism of helminth killing prior to expulsion remains unclear. Here we identify epithelial-cell-derived phospholipase A2 group 1B (PLA2g1B) as a host-derived endogenous anthelmintic. PLA2g1B is elevated in resistant mice and is responsible for killing tissue-embedded larvae. Despite comparable activities of other essential type-2-dependent immune mechanisms, Pla2g1b-/- mice failed to expel the intestinal helminths Heligmosomoides polygyrus or Nippostrongylus brasiliensis. Expression of Pla2g1b by epithelial cells was dependent upon intestinal microbiota, adaptive immunity, and common-gamma chain-dependent signaling. Notably, Pla2g1b was downregulated in susceptible mice and inhibited by IL-4R-signaling in vitro, uncoupling parasite killing from expulsion mechanisms. Resistance was restored in Pla2g1b-/- mice by treating infective H. polygyrus L3 larvae with PLA2g1B, which reduced larval phospholipid abundance. These findings uncover epithelial-cell-derived Pla2g1b as an essential mediator of helminth killing, highlighting a previously overlooked mechanism of anti-helminth immunity

Transcriptomics identified a critical role for Th2 cell-intrinsic miR-155 in mediating allergy and antihelminth immunity

Allergic diseases, orchestrated by hyperactive CD4^(+) Th2 cells, are some of the most common global chronic diseases. Therapeutic intervention relies upon broad-scale corticosteroids with indiscriminate impact. To identify targets in pathogenic Th2 cells, we took a comprehensive approach to identify the microRNA (miRNA) and mRNA transcriptome of highly purified cytokine-expressing Th1, Th2, Th9, Th17, and Treg cells both generated in vitro and isolated ex vivo from allergy, infection, and autoimmune disease models. We report here that distinct regulatory miRNA networks operate to regulate Th2 cells in house dust mite-allergic or helminth-infected animals and in vitro Th2 cells, which are distinguishable from other T cells. We validated several miRNA (miR) candidates (miR-15a, miR-20b, miR-146a, miR-155, and miR-200c), which targeted a suite of dynamically regulated genes in Th2 cells. Through in-depth studies using miR-155^(−/−) or miR-146a^(−/−) T cells, we identified that T-cell–intrinsic miR-155 was required for type-2 immunity, in part through regulation of S1pr1, whereas T-cell–intrinsic miR-146a was required to prevent overt Th1/Th17 skewing. These data identify miR-155, but not miR-146a, as a potential therapeutic target to alleviate Th2-medited inflammation and allergy

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease

IFNγ and IL-12 restrict Th2 responses during Helminth/Plasmodium co-infection and promote IFNγ from Th2 cells

Parasitic helminths establish chronic infections in mammalian hosts. Helminth/Plasmodium co-infections occur frequently in endemic areas. However, it is unclear whether Plasmodium infections compromise anti-helminth immunity, contributing to the chronicity of infection. Immunity to Plasmodium or helminths requires divergent CD4+ T cell-driven responses, dominated by IFNγ or IL-4, respectively. Recent literature has indicated that Th cells, including Th2 cells, have phenotypic plasticity with the ability to produce non-lineage associated cytokines. Whether such plasticity occurs during co-infection is unclear. In this study, we observed reduced anti-helminth Th2 cell responses and compromised anti-helminth immunity during Heligmosomoides polygyrus and Plasmodium chabaudi co-infection. Using newly established triple cytokine reporter mice (Il4gfpIfngyfpIl17aFP635), we demonstrated that Il4gfp+ Th2 cells purified from in vitro cultures or isolated ex vivo from helminth-infected mice up-regulated IFNγ following adoptive transfer into Rag1-/- mice infected with P. chabaudi. Functionally, Th2 cells that up-regulated IFNγ were transcriptionally re-wired and protected recipient mice from high parasitemia. Mechanistically, TCR stimulation and responsiveness to IL-12 and IFNγ, but not type I IFN, was required for optimal IFNγ production by Th2 cells. Finally, blockade of IL-12 and IFNγ during co-infection partially preserved anti-helminth Th2 responses. In summary, this study demonstrates that Th2 cells retain substantial plasticity with the ability to produce IFNγ during Plasmodium infection. Consequently, co-infection with Plasmodium spp. may contribute to the chronicity of helminth infection by reducing anti-helminth Th2 cells and converting them into IFNγ-secreting cells

Elevated Ccl24 from <i>Map3k8</i>^{<i>–/–</i>}mice mediates resistance to <i>H</i>. <i>polygyrus</i> infection.

<p>A) Ratios of ratio plot depicting differentially expressed genes in the duodenal tissue of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. The x axis represents fold change for gene differentially expressed in infected <i>Map3k8</i>^{<i>–/–</i>}mice over infected WT mice and the y axis represents fold change for gene differentially expressed in infected WT or <i>Map3k8</i>^{<i>–/–</i>}mice relative to their respective naïve uninfected controls. B) Top 10 gene regulatory pathways upregulated in D5 <i>H</i>. <i>polygyrus</i> infected <i>Map3k8</i>^{<i>–/–</i>}/ WT mice gut tissue relative to naive control mice as predicted by ingenuity pathway analysis (IPA). The microarray analysis was performed on duodenal tissue from 3 biological replicate samples/genotype for each group.</p

<i>Map3k8</i>^{<i>–/–</i>}mice are resistant to <i>H</i>. <i>polygyrus</i> infection.

<p>A) WT and <i>Map3k8</i>^{<i>–/–</i>}mice were infected with 200 L3 stage <i>H</i>. <i>polygyrus</i> larvae. Adult luminal worms from the intestinal tissue were counted on days 14 (D14) and 28 (D28). B) Fecal egg burden in <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice at D14 and D28. C) ATP levels of adult <i>H</i>. <i>polygyrus</i> worms harvested from the duodenal tissue of WT and <i>Map3k8</i>^{<i>–/–</i>}mice at day 14 post infection. D) Representative FACS plots from the mesenteric lymph nodes (mLNs) and frequency of CD4⁺ <i>Il4</i>^GFP+ and CD4⁺ <i>FoxP3</i>^RFP+ T cells from the spleen, mLNs and Peyer’s patches (PP) of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. E) Total number of cells from the spleen, mLNs and PP of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. F) Arbitrary units (a.u.) of <i>H</i>. <i>polygyrus</i> adult worm extract (HEX)-specific IgG1 in the serum of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. G) HEX-specific IL-5 and IL-13 in mLN cell culture supernatants of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. Data from readouts in A) and B) represent 7–8 mice/genotype pooled from 2 independent experiments. Data from C)—G) is representative of 2–3 independent experiments with 3–4 mice/genotype and 4 adult worms/mouse. * and ** denote p≤0.05 using a two-tailed Mann-Whitney test.</p