Original Research Full Report: Basic and Translational—Alimentary Tract| Volume 160, ISSUE 5, P1679-1693, April 01, 2021

Single-Cell Transcriptional Survey of Ileal-Anal Pouch Immune Cells From Ulcerative Colitis Patients

Published:December 21, 2020DOI:

      Background & Aims

      Restorative proctocolectomy with ileal pouch–anal anastomosis is a surgical procedure in patients with ulcerative colitis refractory to medical therapies. Pouchitis, the most common complication, is inflammation of the pouch of unknown etiology. To define how the intestinal immune system is distinctly organized during pouchitis, we analyzed tissues from patients with and without pouchitis and from patients with ulcerative colitis using single-cell RNA sequencing (scRNA-seq).


      We examined pouch lamina propria CD45+ hematopoietic cells from intestinal tissues of ulcerative colitis patients with (n = 15) and without an ileal pouch–anal anastomosis (n = 11). Further in silico meta-analysis was performed to generate transcriptional interaction networks and identify biomarkers for patients with inflamed pouches.


      In addition to tissue-specific signatures, we identified a population of IL1B/LYZ+ myeloid cells and FOXP3/BATF+ T cells that distinguish inflamed tissues, which we further validated in other scRNA-seq datasets from patients with inflammatory bowel disease (IBD). Cell-type–specific transcriptional markers obtained from scRNA-seq was used to infer representation from bulk RNA sequencing datasets, which further implicated myeloid cells expressing IL1B and S100A8/A9 calprotectin as interacting with stromal cells, and Bacteroidales and Clostridiales bacterial taxa. We found that nonresponsiveness to anti-integrin biologic therapies in patients with ulcerative colitis was associated with the signature of IL1B+/LYZ+ myeloid cells in a subset of patients.


      Features of intestinal inflammation during pouchitis and ulcerative colitis are similar, which may have clinical implications for the management of pouchitis. scRNA-seq enables meta-analysis of multiple studies, which may facilitate the identification of biomarkers to personalize therapy for patients with IBD.
      The processed single cell count tables are provided in Gene Expression Omnibus; GSE162335. Raw sequence data are not public and are protected by controlled-access for patient privacy.


      Abbreviations used in this paper:

      CD (Crohn’s disease), DC (dendritic cell), DE (differential expression), FAP (familial adenomatous polyposis), FDA (Food and Drug Administration), IBD (inflammatory bowel disease), IL (interleukin), IPAA (ileal pouch–anal anastomosis), ISCORE (composite inflammation score), PCA (principal component analysis), PDAI (pouchitis disease activity index), scRNA-seq (single-cell RNA sequencing), TNF (tumor necrosis factor), UC (ulcerative colitis)
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        • Shen B.
        Acute and chronic pouchitis—pathogenesis, diagnosis and treatment.
        Nat Rev Gastroenterol Hepatol. 2012; 9: 323-333
        • Dalal R.L.
        • Shen B.
        • Schwartz D.A.
        Management of pouchitis and other common complications of the pouch.
        Inflamm Bowel Dis. 2018; 24: 989-996
        • Smillie C.S.
        • Biton M.
        • Ordovas-Montanes J.
        • et al.
        Intra- and inter-cellular rewiring of the human colon during ulcerative colitis.
        Cell. 2019; 178: 714-730.e22
        • Mitsialis V.
        • Wall S.
        • Liu P.
        • et al.
        Single-cell analyses of colon and blood reveal distinct immune cell signatures of ulcerative colitis and Crohn’s disease.
        Gastroenterology. 2020; 159: 591-608.e10
        • Haber A.L.
        • Biton M.
        • Rogel N.
        • et al.
        A single-cell survey of the small intestinal epithelium.
        Nature. 2017; 551: 333-339
        • Parikh K.
        • Antanaviciute A.
        • Fawkner-Corbett D.
        • et al.
        Colonic epithelial cell diversity in health and inflammatory bowel disease.
        Nature. 2019; 567: 49-55
        • Corridoni D.
        • Chapman T.
        • Antanaviciute A.
        • et al.
        Inflammatory bowel disease through the lens of single-cell rna-seq technologies. Inflamm Bowel Dis.
        (Available at:)
        • Martin J.C.
        • Chang C.
        • Boschetti G.
        • et al.
        Single-cell analysis of Crohn’s disease lesions identifies a pathogenic cellular module associated with resistance to anti-TNF therapy.
        Cell. 2019; 178: 1493-1508.e20
        • Xu M.
        • Pokrovskii M.
        • Ding Y.
        • et al.
        c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.
        Nature. 2018; 554: 373-377
        • Sano T.
        • Huang W.
        • Hall J.A.
        • et al.
        An IL-23R/IL-22 circuit regulates epithelial serum amyloid A to promote local effector Th17 responses.
        Cell. 2015; 163: 381-393
        • Furusawa Y.
        • Obata Y.
        • Fukuda S.
        • et al.
        Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells.
        Nature. 2013; 504: 446-450
        • Kim K.S.
        • Hong S.-W.
        • Han D.
        • et al.
        Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine.
        Science. 2016; 351: 858-863
        • Esterházy D.
        • Canesso M.C.C.
        • Mesin L.
        • et al.
        Compartmentalized gut lymph node drainage dictates adaptive immune responses.
        Nature. 2019; 569: 126-130
        • Habtezion A.
        • Nguyen L.P.
        • Hadeiba H.
        • et al.
        Leukocyte trafficking to the small intestine and colon.
        Gastroenterology. 2016; 150: 340-354
        • Huang Y.
        • Dalal S.
        • Antonopoulos D.
        • et al.
        Early transcriptomic changes in the ileal pouch provide insight into the molecular pathogenesis of pouchitis and ulcerative colitis.
        Inflamm Bowel Dis. 2017; 23: 366-378
        • Schroeder K.W.
        • Tremaine W.J.
        • Ilstrup D.M.
        Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis.
        N Engl J Med. 1987; 317: 1625-1629
        • Sandborn W.J.
        • Tremaine W.J.
        • Batts K.P.
        • et al.
        Pouchitis after ileal pouch-anal anastomosis: a pouchitis disease activity index.
        Mayo Clinic Proc. 1994; 69: 409-415
        • Heuschen U.A.
        • Allemeyer E.H.
        • Hinz U.
        • et al.
        Diagnosing pouchitis.
        Dis Colon Rectum. 2002; 45: 776-786
        • Zheng G.X.Y.
        • Terry J.M.
        • Belgrader P.
        • et al.
        Massively parallel digital transcriptional profiling of single cells.
        Nat Commun. 2017; 8: 14049
        • Bankhead P.
        • Loughrey M.B.
        • Fernández J.A.
        • et al.
        QuPath: Open source software for digital pathology image analysis.
        Sci Rep. 2017; 7: 16878
        • Dowsett M.
        • Nielsen T.O.
        • A’Hern R.
        • et al.
        Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer Working Group.
        J Natl Cancer Inst. 2011; 103: 1656-1664
        • Stuart T.
        • Butler A.
        • Hoffman P.
        • et al.
        Comprehensive integration of single-cell data.
        Cell. 2019; 177: 1888-1902.e21
        • Lee H.-O.
        • Hong Y.
        • Etlioglu H.E.
        • et al.
        Lineage-dependent gene expression programs influence the immune landscape of colorectal cancer.
        Nat Genet. 2020; 52: 594-603
        • Senda T.
        • Dogra P.
        • Granot T.
        • et al.
        Microanatomical dissection of human intestinal T-cell immunity reveals site-specific changes in gut-associated lymphoid tissues over life.
        Mucosal Immunol. 2019; 12: 378-389
        • Mowat A.M.
        • Agace W.W.
        Regional specialization within the intestinal immune system.
        Nat Rev Immunol. 2014; 14: 667-685
        • Bain C.C.
        • Mowat A.M.
        Macrophages in intestinal homeostasis and inflammation.
        Immunol Rev. 2014; 260: 102-117
        • Fontana R.
        • Raccosta L.
        • Rovati L.
        • et al.
        Nuclear receptor ligands induce TREM-1 expression on dendritic cells: analysis of their role in tumors.
        OncoImmunology. 2019; 8: 1554967
        • West N.R.
        • Hegazy A.N.
        • Owens B.M.J.
        • et al.
        Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor–neutralizing therapy in patients with inflammatory bowel disease.
        Nat Med. 2017; 23: 579-589
        • Mertins P.
        • Mani D.R.
        • Ruggles K.V.
        • et al.
        Proteogenomics connects somatic mutations to signalling in breast cancer.
        Nature. 2016; 534: 55-62
        • Morgan X.C.
        • Kabakchiev B.
        • Waldron L.
        • et al.
        Associations between host gene expression, the mucosal microbiome, and clinical outcome in the pelvic pouch of patients with inflammatory bowel disease.
        Genome Biol. 2015; 16: 67
        • Ramilowski J.A.
        • Goldberg T.
        • Harshbarger J.
        • et al.
        A draft network of ligand–receptor-mediated multicellular signalling in human.
        Nat Commun. 2015; 6: 7866
        • Efremova M.
        • Vento-Tormo M.
        • Teichmann S.A.
        • et al.
        CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes.
        Nat Protoc. 2020; 15: 1484-1506
        • Friedrich M.
        • Pohin M.
        • Powrie F.
        Cytokine networks in the pathophysiology of inflammatory bowel disease.
        Immunity. 2019; 50: 992-1006
        • Arijs I.
        • Hertogh G.D.
        • Lemmens B.
        • et al.
        Effect of vedolizumab (anti-α4β7-integrin) therapy on histological healing and mucosal gene expression in patients with UC.
        Gut. 2018; 67: 43-52
        • Tew G.W.
        • Hackney J.A.
        • Gibbons D.
        • et al.
        Association between response to etrolizumab and expression of integrin αE and granzyme A in colon biopsies of patients with ulcerative colitis.
        Gastroenterology. 2016; 150: 477-487.e9
        • Telesco S.E.
        • Brodmerkel C.
        • Zhang H.
        • et al.
        Gene expression signature for prediction of golimumab response in a Phase 2a open-label trial of patients with ulcerative colitis.
        Gastroenterology. 2018; 155: 1008-1011.e8
        • Ferrante M.
        • D’Haens G.
        • Dewit O.
        • et al.
        Efficacy of infliximab in refractory pouchitis and Crohn’s disease-related complications of the pouch: a Belgian case series.
        Inflamm Bowel Dis. 2010; 16: 243-249
        • Barreiro-de Acosta M.
        • García-Bosch O.
        • Souto R.
        • et al.
        Efficacy of infliximab rescue therapy in patients with chronic refractory pouchitis: a multicenter study.
        Inflamm Bowel Dis. 2012; 18: 812-817
        • Barreiro-de Acosta M.
        • García-Bosch O.
        • Gordillo J.
        • et al.
        Efficacy of adalimumab rescue therapy in patients with chronic refractory pouchitis previously treated with infliximab: a case series.
        Eur J Gastroenterol Hepatol. 2012; 24: 756-758
        • Bär F.
        • Kühbacher T.
        • Dietrich N.A.
        • et al.
        Vedolizumab in the treatment of chronic, antibiotic-dependent or refractory pouchitis.
        Aliment Pharmacol Ther. 2018; 47: 581-587
        • Ollech J.E.
        • Rubin D.T.
        • Glick L.
        • et al.
        Ustekinumab Is Effective for the Treatment of Chronic Antibiotic-Refractory Pouchitis.
        Dig Dis Sci. 2019; 64: 3596-3601
        • Sinha S.R.
        • Haileselassie Y.
        • Nguyen L.P.
        • et al.
        Dysbiosis-induced secondary bile acid deficiency promotes intestinal inflammation.
        Cell Host Microbe. 2020; 27: 659-670.e5
        • Campbell C.
        • McKenney P.T.
        • Konstantinovsky D.
        • et al.
        Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells.
        Nature. 2020; 581: 475-479
        • Hang S.
        • Paik D.
        • Yao L.
        • et al.
        Bile acid metabolites control T H 17 and T reg cell differentiation.
        Nature. 2019; 576: 143-148
        • Song X.
        • Sun X.
        • Oh S.F.
        • et al.
        Microbial bile acid metabolites modulate gut RORγ + regulatory T cell homeostasis.
        Nature. 2020; 577: 410-415
        • Arpaia N.
        • Campbell C.
        • Fan X.
        • et al.
        Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation.
        Nature. 2013; 504: 451-455
        • Schulthess J.
        • Pandey S.
        • Capitani M.
        • et al.
        The short chain fatty acid butyrate imprints an antimicrobial program in macrophages.
        Immunity. 2019; 50: 432-445.e7
        • Kim Y.-G.
        • Kamada N.
        • Shaw M.H.
        • et al.
        The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes.
        Immunity. 2011; 34: 769-780
        • Ramanan D.
        • Tang M.S.
        • Bowcutt R.
        • et al.
        Bacterial sensor Nod2 prevents small intestinal inflammation by restricting the expansion of the commensal Bacteroides vulgatus.
        Immunity. 2014; 41: 311-324
        • Schirmer M.
        • Garner A.
        • Vlamakis H.
        • et al.
        Microbial genes and pathways in inflammatory bowel disease.
        Nat Rev Microbiol. 2019; 17: 497-511
        • Matsuzawa-Ishimoto Y.
        • Hwang S.
        • Cadwell K.
        Autophagy and Inflammation.
        Ann Rev Immunol. 2018; 36: 73-101
        • Marchiando A.M.
        • Ramanan D.
        • Ding Y.
        • et al.
        A Deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection.
        Cell Host Microbe. 2013; 14: 216-224
        • Martin P.K.
        • Marchiando A.
        • Xu R.
        • et al.
        Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota.
        Nat Microbiol. 2018; 3: 1131-1141
        • Neil J.A.
        • Matsuzawa-Ishimoto Y.
        • Kernbauer-Hölzl E.
        • et al.
        IFN-I and IL-22 mediate protective effects of intestinal viral infection.
        Nat Microbiol. 2019; 4: 1737-1749
        • Vasanthakumar A.
        • Moro K.
        • Xin A.
        • et al.
        The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue–resident regulatory T cells.
        Nat Immunol. 2015; 16: 276-285
        • Wang C.
        • Thangamani S.
        • Kim M.
        • et al.
        BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid.
        J Exp Med. 2013; 210: 475-489
        • Luoma A.M.
        • Suo S.
        • Williams H.L.
        • et al.
        Molecular pathways of colon inflammation induced by cancer immunotherapy. Cell 2020;182:655–671.e22.
        (Available at:)
        • Leung J.M.
        • Davenport M.
        • Wolff M.J.
        • et al.
        IL-22-producing CD4+ cells are depleted in actively inflamed colitis tissue.
        Mucos Immunol. 2014; 7: 124-133
        • Tang M.S.
        • Bowcutt R.
        • Leung J.M.
        • et al.
        integrated analysis of biopsies from inflammatory bowel disease patients identifies SAA1 as a link between mucosal microbes with TH17 and TH22 cells.
        Inflamm Bowel Dis. 2017; 23: 1544-1554
        • Lee J.-Y.
        • Hall J.A.
        • Kroehling L.
        • et al.
        Serum amyloid A proteins induce pathogenic Th17 Cells and promote inflammatory disease.
        Cell. 2020; 180: 79-91.e16
        • Ip W.K.E.
        • Hoshi N.
        • Shouval D.S.
        • et al.
        Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages.
        Science. 2017; 356: 513-519
        • Shouval D.S.
        • Biswas A.
        • Kang Y.H.
        • et al.
        Interleukin 1β mediates intestinal inflammation in mice and patients with interleukin 10 receptor deficiency.
        Gastroenterology. 2016; 151: 1100-1104
        • Salas A.
        • Hernandez-Rocha C.
        • Duijvestein M.
        • et al.
        JAK–STAT pathway targeting for the treatment of inflammatory bowel disease.
        Nat Rev Gastroenterol Hepatol. 2020; 17: 323-337
        • Ritchie M.E.
        • Phipson B.
        • Wu D.
        • et al.
        limma powers differential expression analyses for RNA-sequencing and microarray studies.
        Nucleic Acids Res. 2015; 43: e47