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

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

A Novel Role for IκBζ in the Regulation of IFNγ Production

IκBζ is a novel member of the IκB family of NFκB regulators, which modulates NFκB activity in the nucleus, rather than controlling its nuclear translocation. IκBζ is specifically induced by IL-1β and several TLR ligands and positively regulates NFκB-mediated transcription of genes such as IL-6 and NGAL as an NFκB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1β and IL-18, strongly synergize with TNFα for IFNγ production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNγ production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IκBζ protein expression in KG-1 cells upon stimulation with IL-1β, IL-18 and TNFα. Our results demonstrated that IL-18, as well as IL-1β, induced moderate IκBζ expression in KG-1 cells. However, TNFα synergized with IL-1β and IL-18, whereas by itself it had a minimal effect on IκBζ expression. NFκB inhibition resulted in decreased IL-1β/IL-18/TNFα-stimulated IFNγ release. Moreover, silencing of IκBζ expression led to a specific decrease in IFNγ production. Overall, our data suggests that IκBζ positively regulates NFκB-mediated IFNγ production in KG-1 cells

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

Silencing of IκBζ expression suppresses IFNγ and IL-6, not IL-8, mRNA and protein production.

<p>KG-1 cells (2×10⁶/ml) were nucleofected with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IκBζ or 3 different scrambled siRNA oligonucleotides. After 2 h, cells were stimulated with a combination of rIL-1β, rIL-18 and rTNFα (10 ng/ml each) for 24 h. Cells were lysed for mRNA extraction. Messenger RNA (mRNA) was converted to cDNA, followed by quantitative PCR (qPCR) using primers specific for IFNγ (A), IL-6 (B) and IL-8 (C). Supernatants were harvested and analyzed for cytokine release by IFNγ (D), IL-6 (E) and IL-8 (F) ELISA. Results are shown as mean±S.E.M. *, <i>p</i><.05; **, <i>p</i><0.005 (A, B and C, n = 3) (D, E and F, n = 5).</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

Effect of TNFα treatment on KG-1 IL-18R and IL-1R surface expression.

<p>KG-1 cells (10⁶/ml) were incubated with the indicated combinations of rIL-1β, rIL-18, rTNFα (10 ng/ml each) for 24 h. Cells were stained with IL-18R-PE and IL-1R-FITC followed by flow cytometry analysis. Results are representative of 3 separate experiments.</p

The synergistic effect of TNFα and IL-1β/IL-18 on IκBζ protein expression is partially suppressed with a TNFα-specific Ab, but completely blocked with IL-1β and/or IL-18 neutralization.

<p>KG-1 cells (10⁶/ml) were stimulated with rIL-1β, rIL-18, rTNFα (10 ng/ml each) (A), or the indicated combinations of these cytokines (B and C) for the indicated time points. At selected time points, the cells were incubated with the recombinant proteins in the presence of IL-1ra (100 µg/ml), IL-18R Ab (10 µg/ml), TNFα Ab (10 µg/ml), or different combinations of these neutralizing agents. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against IκBζ and actin Ab. Results are representative of at least 3 separate experiments.</p

Silencing of IκBζ expression.

<p>KG-1 cells (2×10⁶/ml) were nucleofected with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IκBζ or 3 different scrambled siRNA oligonucleotides. After 2 h, cells were stimulated with a combination of rIL-1β, rIL-18 and rTNFα (10 ng/ml each) for the indicated time points. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against IκBζ and actin Ab. Results are representative of 5 separate experiments.</p