TMEM258 Is a Component of the Oligosaccharyltransferase Complex Controlling ER Stress and Intestinal Inflammation

Summary - Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5–61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis.Leona M. and Harry B. Helmsley Charitable Trust (2014PG-IBD016)Crohn's and Colitis Foundation of AmericaNational Institutes of Health (U.S.) (grant DK043351)National Institutes of Health (U.S.) (grant DK097485

Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity

<div><p>Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 –which optimizes toll-like receptor signaling from phagosomes–sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to <i>Salmonella</i> Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1β and IL-18 in response to particulate stimuli <i>in vitro</i>, but caspase-1 and IL-1β levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1β, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients.</p></div

AP-3 is required for optimal transcriptional activation of pro-IL-1β and some NLRs after particulate LPS priming.

<p>BMDCs from WT or pearl (pe) mice were untreated or stimulated with LPS or LPS beads for 2 h (<b>A</b>-<b>C</b>) or 3 h (<b>D</b>, <b>E</b>). <b>A-C</b>. cDNA generated from isolated RNA was analyzed by RT-PCR. Shown are mRNA levels of: <b>A</b>, NLRC1, NLRC2, NLRP3; <b>B</b>, pro-IL-1β, pro-IL-18; and <b>C</b>, NLRC4, pro-caspase-1 and ASC. Data from three independent experiments were normalized to the average of five housekeeping genes, and the ΔΔCt values were calculated and represented as mean ± SD fold induction of mRNA in stimulated cells relative to unstimulated cells. <b>D</b>, <b>E.</b> Cell pellets were lysed and fractionated by SDS-PAGE, and NLRP3, NLRC4, pro-caspase-1 (pro-casp. 1), pro-IL-1β and ASC were detected by immunoblotting. <b>D</b>, representative blots. <b>E</b>, quantification of band intensities represented as mean ± SD fold induction in stimulated cells relative to unstimulated cells for pro-IL-1β (<i>top</i>) and NLRP3 (<i>bottom</i>), normalized to tubulin levels. Two or more fold induction was considered significant. *p< 0.05; **p<0.01. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s008" target="_blank">S1 Table</a></b>.</p

AP-3 limits inflammasome sequestration and autophagy induction after STm infection or alum stimulation.

<p><b>(A</b>, <b>B)</b>. WT and pearl (pe) BMDCs expressing ASC-GFP were infected with flagellin-expressing STm (stimulates NLRC4) for 30 or 60 min. Cells were then permeabilized for 1 min with 50 μg/ml digitonin or throughout labeling with 0.1% saponin as indicated, washed, and incubated with mouse anti-GFP and allophycocyanin (APC)-conjugated anti-mouse antibodies. Cells were analyzed by flow cytometry, gating on GFP⁺ cells (R1). <b>A</b>. Shown are representative dot plots of transduced WT and pe DCs indicating gated region based on GFP fluorescence and side scatter (SSC, <i>left panels</i>), and representative histogram plots indicating GFP (<i>middle panels</i>) or APC (anti-GFP) fluorescence (<i>right panels</i>). <i>Black lines</i>, WT; <i>blue lines</i>, pe; <i>dotted lines</i>, secondary antibody alone. <b>B</b>. The ratio of mean fluorescence intensity (MFI) values for anti-GFP signal in digitonin-treated DCs relative to saponin-treated DCs is shown from 4 independent experiments. (<b>C-L)</b>. WT and pearl (pe) BMDCs that were non-transduced (-) or transduced with lentiviruses encoding non-target (ctrl) or either of two ATG7-specific shRNAs or ATG-5- or LC3b-specific shRNAs were infected with STm (<b>C-E</b>) or primed for 3 h with soluble LPS and stimulated with alum (<b>F-L</b>). (<b>C-E</b>) Cell supernatants collected 2 h after Stm infection were assayed for IL-1β by ELISA. <b>C.</b> Representative experiment. <b>D</b>. Data from 3 independent experiments were normalized to IL-1β values from cells treated with non-target shRNA and presented as fold induction. <b>E</b>. IL-1β values for pearl BMDC treated with non-target or ATG7 shRNAs from 3 independent experiments are shown as percent of values for WT DCs treated with the same shRNAs. (<b>F-L</b>) Cell pellets collected 4 h after alum stimulation were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 and tubulin. (<b>F, H</b>). Representative immunoblots, showing pro-caspase-1 (pro-casp.-1) and mature p10 (casp.-1 p10) bands. (<b>G, I</b>) Quantification of band intensities for caspase-1 p10 normalized to pro-caspase-1 and tubulin from three independent experiments are shown as caspase-1 fold change relative to unstimulated (-) WT cells (mean ± SD). (<b>J, K</b>) Data from three independent experiments were normalized to caspase-1 values from untreated cells and presented as fold increase. (<b>L</b>). Caspase-1 values for pearl BMDC treated with non-target, ATG5, ATG7 or LC3b shRNAs from 3 independent experiments are shown as percent of values for WT DCs treated with the same shRNAs. Data in all panels represent mean ± SD. **p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s007" target="_blank">S7 Fig</a></b>.</p

AP-3 promotes survival upon <i>Salmonella</i> Typhimurium infection.

<p>WT CD57BL/6J and congenic pearl (pe) mice were infected orally with 10⁸ STm (+ STm) or received PBS as a control (naïve). (<b>A, B).</b> Mouse survival was assessed over 12 days (<b>A</b>; n = 5) or 7 days (<b>B</b>; n = 11 each mouse type; surviving mice were euthanized on day 7). (<b>C-E).</b> Peyer patches, MLN and spleens were harvested 5 days post-infection, homogenized and plated to measure bacterial load. CFUs were normalized to tissue weight (expressed as CFU/ g of tissue). Data are pooled from three independent experiments. Dotted lines, background (threshold value from uninfected mice); solid lines, geometric means of values above background. *p<0.05; **p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s002" target="_blank">S2</a> Figs</b>.</p

AP-3 is required for perinuclear inflammasome positioning and limits autophagy induction after <i>Salmonella</i> Typhimurium infection in DCs.

<p><b>(A-C).</b> WT and pearl (pe) BMDCs expressing ASC-GFP were infected with flagellin-expressing mCherry-STm (stimulates NLRC4). Cells were fixed 1 h after infection, labeled with DAPI and analyzed by fluorescence microscopy. <b>A.</b> Representative images showing ASC speck (green) relative to STm (red) and nucleus (blue) in four infected WT and pearl DCs each. <b>B.</b> Quantification of perinuclear (within a radius of one μm from the nucleus) and non-perinuclear ASC specks in 20 cells per cell type in each of four independent experiments. <b>C.</b> Quantification of ASC speck distance to the nucleus in 15 cells per cell type in each of three independent experiments. <b>(D</b>, <b>E)</b>. WT and pearl BMDCs were infected with STm, and endogenous LC3 (and actin as a control) was detected by immunoblotting fractionated cell lysates at the indicated time points. <b>D.</b> Representative blot with positions of molecular weight markers (MW) indicated at left. <b>E</b>. Quantification of LC3-II band intensities from three independent experiments, expressed as fold increase relative to unstimulated cells and normalized to LC3-I and β-actin levels. (<b>F</b>-<b>I).</b> WT and pearl BMDCs expressing ASC-GFP alone or with mCherry-LC3 were infected with STm and analyzed by live fluorescence imaging (for LC3) or fixed immunofluorescence microscopy (for p62) 1 h later. <b>F.</b> Representative images showing ASC speck (green) and either LC3 puncta (red, <i>left panels</i>) or endogenous p62 puncta (red) and nuclei (blue; <i>right panels</i>) in infected cells. Corresponding DIC images show nuclear position. <b>G, H.</b> Quantification of LC3 (<b>G</b>) or p62 (<b>H</b>) puncta within a radius of 0.5 μm from the ASC speck (representative image shown at right) in 15 cells per cell type in each of 3 independent experiments. <b>I</b>, Quantification of total p62 puncta normalized to cell area. Data represent mean ± SD. Scale bar: 10 μm.**p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s005" target="_blank">S5</a></b>and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s006" target="_blank">S6</a> Figs</b>.</p

Transcriptional Atlas of Intestinal Immune Cells Reveals that Neuropeptide α-CGRP Modulates Group 2 Innate Lymphoid Cell Responses

Signaling abnormalities in immune responses in the small intestine can trigger chronic type 2 inflammation involving interaction of multiple immune cell types. To systematically characterize this response, we analyzed 58,067 immune cells from the mouse small intestine by single-cell RNA sequencing (scRNA-seq) at steady state and after induction of a type 2 inflammatory reaction to ovalbumin (OVA). Computational analysis revealed broad shifts in both cell-type composition and cell programs in response to the inflammation, especially in group 2 innate lymphoid cells (ILC2s). Inflammation induced the expression of exon 5 of Calca, which encodes the alpha-calcitonin gene-related peptide (α-CGRP), in intestinal KLRG1+ ILC2s. α-CGRP antagonized KLRG1+ ILC2s proliferation but promoted IL-5 expression. Genetic perturbation of α-CGRP increased the proportion of intestinal KLRG1+ ILC2s. Our work highlights a model where α-CGRP-mediated neuronal signaling is critical for suppressing ILC2 expansion and maintaining homeostasis of the type 2 immune machinery