Differences in Th17 cell frequencies in the LP of IL-15 Tg or KO mice, but not in frequencies of Th1 or Treg cells.

<p>Cells from spleen and small intestine LP were stimulated for four hours with PMA and ionomycin and stained with surface markers CD3 and CD4, followed by intracellular staining of IL-17, IFNγ or Foxp3. <b>A.</b> Higher frequencies of Th17 cells were found in the LP of IL-15 KO mice than matched co-caged WT mice (p < 0.001 by Mann-Whitney). <b>B.</b> IL-15 Tg mice showed significantly lower frequencies of Th17 cells in the LP compared with their co-caged WT littermates (the littermates were considered as matched WT controls) (p < 0.001 by Mann-Whitney). <b>C.</b> The frequencies of IFNγ -producing CD4⁺ T cells were not significantly different in the LP of IL-15 KO <i>vs</i> WT and IL-15 Tg <i>vs</i> littermate WT control. <b>D.</b> The frequencies of CD3⁺CD4⁺Foxp3⁺ cells were not significantly different in small intestinal LP of IL-15 KO <i>vs</i> WT and IL-15 Tg <i>vs</i> littermate WT control. This figure pools data from several independent experiments with similar results. <b>D</b>ouble asterisk (**denotes P<0.001).</p

Characterization of MP subsets from intestinal LP and spleen.

<p>MPs were purified from small intestinal LP and spleen and then stained for surface markers CD103, CD11b, CD8, and MHCII. <b>A.</b> Purified MPs from intestinal LP were analyzed for CD11b and CD103 expression. One representative mouse was shown. <b>B.</b> Purified MPs from the spleen were analyzed for CD11b and CD103 expression. Far fewer CD103⁺ MPs were found in the spleen, as expected, but CD103 expression was higher in IL-15 Tg compared to WT mice. Conversely, CD11b expression was slightly lower in IL-15 Tg compared to WT, but slightly higher in the IL-15 KO <i>vs</i> WT. <b>C</b>. MPs purified from LP were analyzed for CD8α and CD103 expression. FACS plots are representative of five independent experiments of two mice pooled in each. The pooled data are shown in D, E and F. D. Percent of double positive CD11b and CD103 MP in the LP. E. Percent of single positive CD103 MPs. F. Percent of single positive CD11b MPs. The ratios of CD11b⁺CD103⁺/ CD103⁺ MPs (G) and CD11b⁺CD103^-/ CD103⁺ MPs (H) based on purified LP cell preparations are shown in G and H. Paired Student t-tests were used for comparing different CD11b, CD103 subset MPs.</p

Flow cytometry of intracellular production of IL-17 by LP MPs from IL-15 Tg (A and B) or IL-15 KO (C and D) cocultured for 4 days with OTII T cells under Th17 conditions and re-stimulated for 4h with PMA and ionomycin.

<p>Data are representative of one of three independent experiments with comparable results. <b>A, B.</b> LP MPs from IL-15 Tg have lower capacity than WT to induce antigen specific Th17 differentiation <i>in vitro</i> (p = 0.0075 from Mann-Whitney test of 5 mice per group shown in B). <b>C, D.</b> LP MPs from IL-15 KO have higher capacity to induce antigen specific Th17 differentiation <i>in vitro</i> (p = 0.038 from Mann-Whitney test of 9 mice per group shown in D). B, D. The fold change of the induction of antigen specific Th17 relative to WT MPs is shown. For normalization, the induction of antigen specific of Th17 by LP MPs from WT was considered as 1 (<b>*</b>P<0.05 for N ≥5). E. Effect of adding or blocking IL-15 in the culture of OT II CD4⁺ T cells with WT LP MPs to induce Th17 cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143001#sec002" target="_blank">Methods</a>. Both anti-mouse IL-15 and anti-human IL-15, which react with mouse IL-15, were tested and are shown in the lower panels.</p

Human mutations in integrator complex subunits link transcriptome integrity to brain development

<div><p>Integrator is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. Importantly, its role in human development and disease is so far largely unexplored. Here, we provide evidence that biallelic <i>Integrator Complex Subunit 1 (INTS1)</i> and <i>Subunit 8</i> (<i>INTS8)</i> gene mutations are associated with rare recessive human neurodevelopmental syndromes. Three unrelated individuals of Dutch ancestry showed the same homozygous truncating <i>INTS1</i> mutation. Three siblings harboured compound heterozygous <i>INTS8</i> mutations. Shared features by these six individuals are severe neurodevelopmental delay and a distinctive appearance. The <i>INTS8</i> family in addition presented with neuronal migration defects (periventricular nodular heterotopia). We show that the first <i>INTS8</i> mutation, a nine base-pair deletion, leads to a protein that disrupts INT complex stability, while the second missense mutation introduces an alternative splice site leading to an unstable messenger. Cells from patients with <i>INTS8</i> mutations show increased levels of unprocessed UsnRNA, compatible with the INT function in the 3’-end maturation of UsnRNA, and display significant disruptions in gene expression and RNA processing. Finally, the introduction of the <i>INTS8</i> deletion mutation in P19 cells using genome editing alters gene expression throughout the course of retinoic acid-induced neural differentiation. Altogether, our results confirm the essential role of Integrator to transcriptome integrity and point to the requirement of the Integrator complex in human brain development.</p></div

Effect of <i>INTS8ΔEVL</i> mutation on P19 cell neuronal differentiation.

<p>(A) Two P19 clonal cell lines bearing a homozygous <i>INTS8ΔEVL</i> mutation were generated by CRISPR/Cas9 mediated genome editing using two different guide RNAs. After genomic DNA extraction, the region surrounding the <i>INTS8ΔEVL</i> mutation is amplified by PCR and the corresponding DNA digested with NheI to detect homologous recombination or mock digested (Unc = uncut). The P19 parental cell line is used as a control. (B) The protein expression of different INT subunits is monitored by Western Blot in total cellular extracts of <i>INTS8ΔEVL</i> mutant P19 cell lines. The P19 parental cell line is used as a control. Tubulin serves as a loading control. (C) Expression of neuronal differentiation markers during RA-induced differentiation of P19 cells. Wild-type and <i>INTS8ΔEVL</i> P19 cell lines are treated with RA and let to differentiate for 8 days. Cells are harvested at the indicated time points after the initiation of the differentiation protocol (D0 = day zero, D2 = day two, D4 = day4, D8 = day8) and RNA was extracted and reverse transcribed. Gene expression is determined by qRT-PCR (n = 3, +/- SEM). <i>GAPDH</i> expression is used as a normalizer.</p

Dysregulated transcriptome in patient skin fibroblasts.

<p><b>(</b>A, B) qRT-PCR validation of gene expression variation in patient cells for two illustrative examples, <i>NPTX1</i> and <i>OSR2</i> mRNAs. (C) Correlation analysis of differential gene expression data from exon arrays (X axis) and RNA-seq (Y axis). (D) Pie chart representing the different types of alternative splicing events detected in patient cells vs control in RNA-seq data (n = 215, p<0.01, 292 total events). (E, F) Experimental verification by RT-PCR of the splicing changes associated with <i>INTS8</i> mutations for two illustrative examples <i>ADAM15</i> (E) and <i>ATL3</i> (F) mRNAs.</p

Characterization of the <i>INTS8</i> mutations.

<p>(A) qRT-PCR on fibroblast-derived RNA of the patients (III-2, III-3, III-4), their unaffected sibling (III-1), and two age-matched control cell lines (C1, C2), normalized for <i>GAPDH</i> expression. Expression of the c.893A>G allele vs. wild type was measured using a primer located at the c.2917-2925del locus (<i>INTS8</i> non-ΔEVL allele). (B) Schematic overview of INTS8 genomic and protein sequence. The c.893A>G mutation (in red) is located at the 5’ end of the exon8 of the transcript variant 3 that contains a premature stop codon (PTC). (C) Schematic of the GFP-minigene reporter construct used to evaluate the effect of the c.893A>G mutation on <i>INTS8</i> exon 8 splicing pattern. Size of the corresponding amplicons is indicated on the left (D) RT-PCR analysis of RNA isolated from HeLa (lanes 1–3) or HEK293T cells (lanes 4–6) transfected with the GFP-minigene constructs. The empty reporter (GFP) is used as a control. (E) Western blot analysis of flag-affinity eluates from HEK293T stable lines expressing 3xFlag-tagged INTS8 wild type (WT) or INTS8ΔEVL. (F) Western blots on total cell extracts from patient and control primary fibroblasts. (G) qRT-PCR showing normalized expression of misprocessed U1, U2 and U4 snRNAs in total RNA extracted from patient III-2 and III-4 fibroblasts compared to two controls. All pairwise comparisons between patient and control UsnRNA levels are significant (at least p<0.05, Student’s T-test) to the exception of III-2 and C1 for UsnRNA U1 (p<0.06).</p

Clinical phenotype of human <i>INTS1</i> and <i>INTS8</i> mutations.

<p>Clinical phenotype of human <i>INTS1</i> and <i>INTS8</i> mutations.</p

Biallelic <i>INTS8</i> mutations in a family with a severe neurodevelopmental syndrome.

<p>(A-C) Magnetic resonance imaging (MRI) of affected individual III-2 showing cerebellar hypoplasia (A,C, arrow), reduced volume of the pons and brainstem and periventricular nodular heterotopia (B, arrows) versus (D-F) normal MRI from unaffected individual. (G) Pedigree of the extended family; filled symbols represent affected individuals. Below each individual the <i>INTS8</i> alleles (wt = wild type) are shown. (H) Schematic of INTS8 including the four tetratricopeptide (TPR) motifs (blue blocks), the patient mutations and in the lower panel the conservation of the affected amino acids residues throughout evolution. (I-L) Electropherograms from Sanger sequencing of <i>INTS8</i> wild type and mutant alleles.</p