Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Stable loss of major histocompatibility complex class I (MHC-I) gene DNA methylation (DNAm) in intestinal epithelial organoids (IEOs) derived from patients with Crohn’s disease (CD). (A) (i) Overview of experimental set-up and sample generation. (ii) Representative brightfield images of IEOs. Scale bars: 300 µm. (B) (i) Correlation heat map of comethylated CpG modules identified by weighted gene coexpression network analysis (WGCNA) in terminal ileum (TI) IEOs. Module 17 (ME17) demonstrates hypomethylation and the strongest association with CD diagnosis (R=−0.43, p value<0.001). (ii) Gene set enrichment analysis performed on module 17, showing a significant loss of DNAm in CD organoids compared with healthy controls and UC in TI. (C) DNAm (beta value) of four representative MHC-I related Differetial Methylated Positions (DMPs) showing CD-associated loss of DNAm in TI and sigmoid colon (SC) but not duodenum (DUO) organoids (DUO=54, TI=127 and SC=131). (D) Average DNAm (beta value) of all CpGs located in MHC-I related genes for IEOs split by diagnosis, gut segment and inflammatory status. (E) (i) Correlation of nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm between early and later passage IEOs from the same individuals including patients diagnosed with CD (blue), UC (yellow) and controls (grey, n=22 patients. (ii) DNAm (beta values) of CpGs located in NLRC5 and TAP1 at high passage (>7) IEOs (cohort 1, n=22). (F) Average MHC-I (i) and NLRC5 (ii) DNAm as well as NLRC5 gene expression (iii) in control patient-derived TI IEOs stimulated with proinflammatory cytokines interferon γ (IFNγ) and tumour necrosis factor α (TNFα) (n=5). (False Discovery Rate (FDR) * < 0.05, FDR **< 0.01, FDR***< 0.001, FDR**** < 0.0001, ns=not significant.)

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Derivative Assay, Biomarker Discovery, Methylation, Binding Assay, Gene Expression, Control

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Loss of major histocompatibility complex class I (MHC-I) DNA methylation (DNAm) correlates with increased gene expression in primary intestinal epithelium of patients with Crohn’s disease (CD). (A) Overview of patient cohort, sample preparation and data generation. (B, C) DNAm and gene expression in purified terminal ileum (TI) (B) and sigmoid colon (SC) (C) epithelium. (i) Average DNAm (beta value) of all and selected MHC-I pathway-related CpGs showing significant, CD-associated loss of DNAm. (ii) Correlation between beta values and corresponding gene expression (R=Spearman’s rank correlation). (D) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm in the IE of healthy, patients with UC and CD at the point of diagnosis and during reassessment. (E) Correlation of NLRC5 promoter DNAm in intestinal epithelial organoids (IEOs) obtained from the same patient at diagnosis and reassessment (Spearman’s rank correlation). (F) NLRC5 promoter DNAm in TI IEOs derived from patients with CD, UC and control (n=3 IEO per condition, two-way analysis of variance (ANOVA) with Turkey’s test for multiple comparisons. ****p<0.0001). (G) NLRC5 mRNA expression in TI IEOs derived from patients with controls, UC and CD at baseline and on interferon γ (IFNγ) treatment (10 ng/mL for 6 hours). Data are normalised to the mean of control lines and shown as mean±SEM (two-way ANOVA with Turkey’s test for multiple comparisons. **P<0.01, *p<0.05, ns=not significant). n=3 IEO lines in each group for three independent experiments.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Gene Expression, Sample Prep, Purification, Binding Assay, Biomarker Discovery, Derivative Assay, Control, Expressing

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acts as transcriptional transactivator of intestinal epithelial cell (IEC) major histocompatibility complex class I (MHC-I) and potentiates the effect of interferon γ (IFNγ). (A) Overview of experimental set-up. (B) Heatmap showing gene expression (RNAseq) of MHC-I pathway genes in terminal ileum (TI) intestinal epithelial organoids (IEOs)± NLRC5 overexpression (dox), and ±exposure to IFNγ (n=4 independent replicates). (C) RNA transcription of HLA-A / -B / -C / -E / -F / -G in response to IFNγ and tumour necrosis factor α (TNFα) in wild type (WT) and NLRC5 OE TI IEOs. (D) Relative expression for MHC-I pathway genes in WT ( NLRC5 +/+ ) and corresponding NLRC5 deficient ( NLRC5 −/ − ) TI IEOs±IFNγ (n=3 replicates. Two-way analysis of variance (ANOVA) with Bonferroni’s test for multiple comparisons, **p<0.01, ***p<0.001, ****p<0.0001). Data are representative of two independent experiments. (E) Immunofluorescence spinning disc microscopy of organoids described in D, ±IFNγ (48 hours). (i) Representative images of untreated (BSA) and treated (IFNγ) WT ( NLRC5 +/+ ) and NLRC5 deficient ( NLRC5 −/ − ) TI IEOs taken by Opera Phoenix. Scale bar=2 mm. (ii) HLA-A,B,C mean intensity quantification of BSA and IFNγ NLRC5 +/+ and NLRC5 −/ − TI IEOs. (n=3 independent replicates. Two-way ANOVA with Bonferroni multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001.) (F) Correlation between mRNA gene expression of NLRC5 and (i) HLA-B and (ii) HLA-E, in purified TI and sigmoid colon (SC) epithelium (cohort 2) (Spearman’s rank correlation).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Binding Assay, Immunopeptidomics, Gene Expression, Over Expression, Expressing, Immunofluorescence, Microscopy, Purification

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Crohn’s disease (CD)-associated increased intestinal epithelial major histocompatibility complex class I (MHC-I) expression affects the stem cell compartment and follows a crypt-villus gradient. (A) Summary of experimental set-up. (B) (i) Schematic representation of intestinal epithelial cell (IEC) subtypes and their location within the small bowel (terminal ileum (TI)) crypt-villus structure (TA—transiently amplifying cells). (ii) Uniform manifold approximation and projection (UMAP) plot demonstrating single IEC transcriptomes present in TI mucosal biopsies obtained from children newly diagnosed with CD and non-IBD controls. (C) Top panel: violin plots showing crypt-villus scores of cells within each identified cell subtype (top left) and total number of cells (top right). Bottom panel: correlation between MHC-I summary score and crypt-villus scores for all IEC transcriptomes. Best fitting correlation is displayed as individual lines for CD (blue), UC (yellow) and non-IBD control samples (grey) (bottom left). Bottom right: box plots of summary MHC-I single-cell transcriptional score split by diagnosis. (D) Summary/average MHC-I score in individual IEC subtypes comparing CD, UC and controls. (E) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) expression in TI IEC of patients with CD colocalises with CD8+ T cells. RNA scope of TI biopsies from healthy donors and patients with CD. EPCAM (cyan), NLRC5 (white), TAP1 (yellow), CD8A (red), IFNG (green) and nuclei (DAPI, blue). Proximity of CD8 + T-cells with NLRC5 + EPCAM + cells in the CD biopsy is shown with arrows. Representative images are shown. Scale bar=100 µm and zoom in scale bar=10 µm.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, Expressing, Control, Biomarker Discovery, Binding Assay, RNAscope

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Intestinal epithelial cells (IECs) present antigen via major histocompatibility complex class I (MHC-I) and activate CD8 + T cells in vitro with nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acting as key modulator of mucosal inflammation in vivo. (A) Overview of experimental set-up. (B) Quantification of H2K b -SIINFEKL and pan-H2K b flow cytometry on live EpCAM + cells in murine intestinal epithelial organoids (IEOs) stimulated with or without interferon γ (IFNγ) (48 hours) and pulsed with or without OVA257–264 peptide (SIINFEKL) peptide. Data are representative of two independent experiments run in triplicates. GMFI, geometric mean fluorescence intensity; AU, arbitrary units. P values were calculated by two-way analysis of variance (ANOVA) with Bonferroni test for multiple comparisons (**p<0.01, ****p<0.0001). (C) Overview of experimental design. (D) Quantitative PCR gene expression of Ifng for coculture experiment in murine IEOs±SIINFEKL peptide pulse and cocultured with SIINFEKL-activated OTI T-cells. Data are presented as fold change over unstimulated OTI cells minus murine IEOs, normalised to Cd8a . P values were calculated using two-way ANOVA with Bonferroni’s multiple comparisons test (***p<0.001, ns=not significant). (E) Body weight changes over time during and after a 6-day course of 2% dextran sulphate sodium (DSS) exposure. (n=8 and n=5 Nlrc5fl/fl and Nlrc5-/- mice, respectively. P values calculated by multiple t-tests with Holm-Šídák correction for multiple comparisons.) (F) Quantification of H2K b surface expression on EpCAM+ cell populations within the lamina propria extractions of DSS-treated mice. All panels: data are representative of two independent experiments (**p<0.01). (G) Colon weight per unit length and mesenteric lymph node (MLN) weight and spleen weight of Nlrc5 wild type and knockout mice, on day 14 after initiation of 6-day course of 2% DSS (**p<0.01).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, In Vitro, Binding Assay, In Vivo, Flow Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Knock-Out

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: NLRC5-dependent activation of the inflammasome

doi: 10.4049/jimmunol.1003111

Figure Lengend Snippet: A) Lentivirus transduced THP-1 cells were stimulated with E. coli. Cell supernants were assayed for IL-1β by ELISA. Two shNLRC5 and their control shRNA with sequence specific mutations (shControl1 and shControl2) were analyzed. B) ShRNA containing Control cell lines were assayed for IL-1β secretion in response to E. coli: shEV = empty vector; shLuc = shRNA to luciferase; mshASC = mutant ASC; mshNLRC5 = mutant shNLRC5#1; shASC, shNLRP3, shNLRC5 = sequence specific shRNA. C) THP-1 cells were nucleofected with siRNAs specific for NLRP3 and NLRC5 and with a non-silencing (siControl) control siRNA, 48 hours cells were stimulated as in (A). D) Human primary monocytes were transfected with siRNA to NLRC5 and a non-silencing siRNA (siControl) and stimulated as in (C). In D) values were normalized to non-silencing control. In A-C) experiments were repeated at least three independent times. Untreated samples were below the level of detection. Asterisks denote p < 0.05. (E) THP-1 cell lysates were probed for active IL-1β in response to E. coli infection and active capase-1 (F).

Article Snippet: Quantitative PCR analysis of NLRC5 expression Human total RNA Master Panel II (Clontech) and mouse tissue RNA was used for cDNA synthesis using standard procedures.

Techniques: Enzyme-linked Immunosorbent Assay, shRNA, Sequencing, Plasmid Preparation, Luciferase, Mutagenesis, Transfection, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: NLRC5-dependent activation of the inflammasome

doi: 10.4049/jimmunol.1003111

Figure Lengend Snippet: A) FLAG™ tagged NLRP3 and ASC were co-transfected with NLRC5-V5. Lysates were immunoprecipitated with α-V5 and probed with α-FLAG™ mAbs. NLRC5 specifically interacts with NLRP3 and ASC. B). Truncation mutants of NLRC5 were co-transfected with NLRP3-FLAG. Lyastes were immunoprecipated with α-V5 mAb and probed with α-FLAG™. C). HEK293T cells were transfected with caspase-1 and IL-1β in the presence of increasing amounts of ASC in the presence or absence of NLRs. Supernatants were analyzed for IL-1β by ELISA. D) HEK293T cells were transfected with increasing concentrations of NLRP3 or NLRC5 in the presence of ASC, pro-caspase-1 and –IL-1β. Cell lysates were probed for cleaved IL-1β.

Article Snippet: Quantitative PCR analysis of NLRC5 expression Human total RNA Master Panel II (Clontech) and mouse tissue RNA was used for cDNA synthesis using standard procedures.

Techniques: Transfection, Immunoprecipitation, Enzyme-linked Immunosorbent Assay