Intestinal dendritic cells specialize to activate transforming growth factor-β and induce Foxp3+ regulatory T cells via integrin αvβ8

BACKGROUND & AIMS: The intestinal immune system is tightly regulated to prevent responses against the many nonpathogenic antigens in the gut. Transforming growth factor (TGF)-β is a cytokine that maintains intestinal homeostasis, in part by inducing Foxp3(+) regulatory T cells (Tregs) that suppress immune responses. TGF-β is expressed at high levels in the gastrointestinal tract as a latent complex that must be activated. However, the pathways that control TGF-β activation in the intestine are poorly defined. We investigated the cellular and molecular pathways that control activation of TGF-β and induction of Foxp3(+) Tregs in the intestines of mice to maintain immune homeostasis. METHODS: Subsets of intestinal dendritic cells (DCs) were examined for their capacity to activate TGF-β and induce Foxp3(+) Tregs in vitro. Mice were fed oral antigen, and induction of Foxp3(+) Tregs was measured. RESULTS: A tolerogenic subset of intestinal DCs that express CD103 were specialized to activate latent TGF-β, and induced Foxp3(+) Tregs independently of the vitamin A metabolite retinoic acid. The integrin αvβ8, which activates TGF-β, was significantly up-regulated on CD103(+) intestinal DCs. DCs that lack expression of integrin αvβ8 had reduced ability to activate latent TGF-β and induce Foxp3(+) Tregs in vitro and in vivo. CONCLUSIONS: CD103(+) intestinal DCs promote a tolerogenic environment in the intestines of mice via integrin αvβ8-mediated activation of TGF-β

Multiregional origins of the domesticated tetraploid wheats

We used genotyping-by-sequencing (GBS) to investigate the evolutionary history of domesticated tetraploid wheats. With a panel of 189 wild and domesticated wheats, we identified 1,172,469 single nucleotide polymorphisms (SNPs) with a read depth ≥3. Principal component analyses (PCAs) separated the Triticum turgidum and Triticum timopheevii accessions, as well as wild T. turgidum from the domesticated emmers and the naked wheats, showing that SNP typing by GBS is capable of providing robust information on the genetic relationships between wheat species and subspecies. The PCAs and a neighbour-joining analysis suggested that domesticated tetraploid wheats have closest affinity with wild emmers from the northern Fertile Crescent, consistent with the results of previous genetic studies on the origins of domesticated wheat. However, a more detailed examination of admixture and allele sharing between domesticates and different wild populations, along with genome-wide association studies (GWAS), showed that the domesticated tetraploid wheats have also received a substantial genetic input from wild emmers from the southern Levant. Taking account of archaeological evidence that tetraploid wheats were first cultivated in the southern Levant, we suggest that a pre-domesticated crop spread from this region to southeast Turkey and became mixed with a wild emmer population from the northern Fertile Crescent. Fixation of the domestication traits in this mixed population would account for the allele sharing and GWAS results that we report. We also propose that feralization of the component of the pre-domesticated population that did not acquire domestication traits has resulted in the modern wild population from southeast Turkey displaying features of both the domesticates and wild emmer from the southern Levant, and hence appearing to be the sole progenitor of domesticated tetraploids when the phylogenetic relationships are studied by methods that assume a treelike pattern of evolution.This work was supported by: TAB, 339941, European Research Council; TAB, NE/M010805/1, Natural Environment Research Councilinfo:eu-repo/semantics/publishedVersio

Diversity of a cytokinin dehydrogenase gene in wild and cultivated barley

The cytokinin dehydrogenase gene HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1–3 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley

Tunisia’s youth: awakened identity and challenges post-Arab Spring

Regulation of an immune response requires complex crosstalk between cells of the innate and adaptive immune systems, via both cell–cell contact and secretion of cytokines. An important cytokine with a broad regulatory role in the immune system is transforming growth factor-β (TGF-β). TGF-β is produced by and has effects on many different cells of the immune system, and plays fundamental roles in the regulation of immune responses during homeostasis, infection and disease. Although many cells can produce TGFβ, it is always produced as an inactive complex that must be activated to bind to the TGFβ receptor complex and promote downstream signalling. Thus, regulation of TGFβ activation is a crucial step in controlling TGFβ function. This review will discuss how TGFβ controls diverse immune responses and how TGFβ function is regulated, with a focus on recent work highlighting a critical role for the integrin αvβ8 expressed by dendritic cells in activating TGFβ

Ablation of enhanced IL-13 production by CD4+ T-cells in <i>Itgb8 (CD11c-Cre)</i> mice restores susceptibility to infection with <i>T. muris</i>.

<p>(<i>A</i>) IL-13 production by LILP CD4+ T-cells isolated day 3 post-infection (p.i.) with a chronic dose of <i>T. muris</i> eggs in IL-4−/− control and <i>Itgb8 (CD11c-Cre)</i> IL-4−/− mice. Representative flow cytometry plots and mean data are displayed. Data (n = 5–6 mice per group) are from two independent experiments. Data (n = 5–6) are from two independent experiments performed. (<i>B</i>) Worm burdens from IL-4−/− and <i>Itgb8 (CD11c-Cre)</i> IL-4−/− mice at day 14 p.i. with a chronic dose of <i>T. muris</i> eggs. Data (n = 5–6 mice per group) are from two independent experiments. **, P<0.01; N.S., not significant via Student's <i>t</i> –test for the indicated comparisons between groups, error bars represent SE of means.</p

Protection from chronic <i>T. muris</i> infection in mice lacking the TGFβ-activating integrin αvβ8 on DCs is dependent on CD4+ T-cells, but does not involve Foxp3+ Tregs.

<p>(<i>A</i>) Worm burdens from control and <i>Itgb8 (CD11c-Cre)</i> mice crossed onto a SCID background analysed at day 32 post-infection (p.i.) with a chronic dose of <i>T.muris</i> eggs. Data (n = 4–9 mice per group) are from two independent experiments performed. (<i>B</i>) Worm burdens from <i>Itgb8 (CD11c-Cre)</i> mice infected with a chronic dose of <i>T.muris</i> eggs and treated with 2 mg of control IgG or anti-CD4 antibody (YTS191) analysed at day 17 p.i. Data (n = 6 mice per group) are from two independent experiments performed. (<i>C</i>) Control and DEREG mice were infected with a chronic dose of <i>T. muris</i> eggs, injected i.p. with 200 ng diphtheria toxin every 2 days (starting 2 days before infection) and worm burdens analysed at day 14 p.i. Data (n = 10 mice per group) are from two independent experiments performed. (<i>D</i>) Percentage of Foxp3+ Tregs of CD4 T-cells in mLN, IEL and LILP populations during <i>T. muris</i> infection as analysed by flow cytometry. Data (n = 7–11 mice per group) are from four independent experiments. (<i>E</i>) Caecal lamina propria worm burdens in control, <i>Itgb8 (CD11c-Cre)</i> and <i>Itgb8 (CD11c-Cre)</i> mice injected with 0.5×10⁶ GFP-Foxp3+ CD4 T-cells 3 days prior to infection. All mice received a chronic dose of <i>T. muris</i> and were analysed at day 14 p.i. for worm burden or day 3 p.i. for reconstitution. Data (n = 3–4 mice per group) are from three independent experiments. **, P<0.01; or ***, P<0.005, N.S., not significant via Kruskal–Wallis (D) and Student's <i>t</i>-test (A, B, C and E) for the indicated comparisons between groups, error bars represent SE of means.</p

Mice lacking the TGFβ-activating integrin αvβ8 on DCs demonstrate an enhanced IL-13 production in LILP CD4+ T-cells.

<p>(<i>A</i>) IL-13 and (<i>B</i>) IFNγ cytokine levels from <i>T. muris</i> E/S antigen-stimulated mLN cells from control and <i>Itgb8 (CD11c-Cre)</i> mice at different time-points post-infection ( p.i.) with a chronic dose of <i>T. muris</i> eggs, determined via cytometric bead array/ELISA. Data (n = 5–6 mice per group) are from three or more independent experiments. (<i>C</i>) Analysis of intracellular IL-13 and IFNγ expression in CD4+ T-cells isolated from control and <i>Itgb8 (CD11c-Cre)</i> mice at day 3 p.i. with a chronic dose of <i>T. muris</i> eggs. Representative flow cytometry plots and mean data are displayed. Data (n = 5–6 mice per group mice per group) are from three independent experiments. (<i>D</i>) Representative flow cytometry plots for IL-13 and IFNγ from LILP CD4+ T-cells at day 3 p.i. and (<i>E</i>) Serum antigen specific IgG1 and IgG2a levels at day 21–23 p.i. from control and anti-TGFβ antibody-treated C57BL/6 mice infected with a chronic dose of <i>T. muris</i> eggs. Data (n = 7–9 mice per group) are from two independent experiments performed. *, P<0.05; **, P<0.01; or ***, P<0.005, N.S., not significant via Kruskal–Wallis (A and B) and Student's <i>t</i>-test (C and E) for the indicated comparisons between groups, error bars represent SE of means.</p

Mice lacking the TGFβ-activating integrin αvβ8 on DCs are protected from a chronic <i>T. muris</i> infection.

<p>(<i>A</i>) Representative flow cytometry plots and (<i>B</i>) average MFI values for p-Smad 2/3 staining in CD4+ T-cells from control and <i>Itgb8 (CD11c-Cre)</i> mice at different times after infection of mice with a chronic dose of <i>T. muris</i> eggs. Data (n = 5–8) are from three independent experiments performed. <i>(C)</i> Western blot analysis of p-Smad2/3 and β-actin in purified CD4+ T-cells from control and <i>Itgb8 (CD11c-Cre)</i> mice at different times during infection with a chronic dose of <i>T. muris</i>. Data representative of two independent experiments. (<i>D</i>) TGFβ activation by intestinal DCs isolated from control or <i>Itgb8 (CD11c-Cre)</i> mice, from naive mice or day 3 post-infection (p.i.) with a chronic dose of <i>T. muris</i> eggs, detected by co-culture with an active TGFβ reporter cell line <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003675#ppat.1003675-Abe1" target="_blank">[15]</a>. Data (n = 3–4) are from three independent experiments performed. <i>(E)</i> Worm burdens from control and <i>Itgb8 (CD11c-Cre)</i> mice at day 14 and 35 p.i. with a chronic dose of <i>T. muris</i> eggs. Data (n = 9–10 mice per group) are from at least two independent experiments performed. *, P<0.05, ***, P<0.005 via Kruskal–Wallis (B) and Student's <i>t</i>-test (D and E) for the indicated comparisons between groups, error bars represent SE of means.</p

TGFβ is functionally important in the development of chronic <i>T. muris</i> infection.

<p>(<i>A</i>) and <i>(B)</i> Analysis of p-Smad 2/3 in CD4+ T-cells from mLN during development of a chronic <i>T. muris</i> infection in C57BL/6 mice. (<i>A</i>) Representative flow cytometry plots in naive mice and mice analysed 3 or 7 days post-infection (p.i.), (<i>B</i>) analysis of p-Smad 2/3 (MFI) in mLN CD4+ T-cells at different timepoints during infection. Data (n = 4–8 mice per group) are from up to three independent experiments performed. <i>(C)</i> Worm burdens from control and anti-TGFβ antibody (clone 1D11)-treated C57BL/6 mice analysed at day 21–23 p.i. after infection with a chronic dose of <i>T. muris</i> eggs. Data (n = 7–9 mice per group) are from two independent experiments performed. *, P<0.05; ***, P<0.005 via Kruskal–Wallis (B) and Student's <i>t</i>-test (C), error bars represent SE of means.</p