Predominance of Type 1 Innate Lymphoid Cells in the Rectal Mucosa of Patients With Non-Celiac Wheat Sensitivity: Reversal After a Wheat-Free Diet

Diana Di Liberto, Pasquale Mansueto, Alberto D'Alcamo, Marianna Lo Pizzo, Elena Lo Presti, Girolamo Geraci, Francesca Fayer, Giuliana Guggino, Giuseppe Iacono, Francesco Dieli, Antonio Carroccio, Diana Di Liberto, Pasquale Mansueto, Alberto D'Alcamo, Marianna Lo Pizzo, Elena Lo Presti, Girolamo Geraci, Francesca Fayer, Giuliana Guggino, Giuseppe Iacono, Francesco Dieli, Antonio Carroccio

Abstract

Objectives: Non-celiac wheat sensitivity (NCWS) is defined as a reaction to ingested wheat after exclusion of celiac disease and wheat allergy. As its pathogenesis is incompletely understood, we evaluated the inflammatory response in the rectal mucosa of patients with well-defined NCWS.

Methods: The prospective study included 22 patients with irritable bowel syndrome (IBS)-like clinical presentation, diagnosed with NCWS by double-blind placebo-controlled challenge. Eight IBS patients not improving on wheat-free diet were used as controls. Two weeks after oral challenge was performed with 80 grams of wheat daily, cells were isolated from rectal biopsies and thoroughly characterized by fluorescence-activated cell sorting analysis for intracellular cytokines and surface markers.

Results: Rectal biopsies from wheat-challenged NCWS patients showed that a significant mucosal CD45(+) infiltrate consisted of CD3(+) and CD3(-) lymphocytes, with the latter spontaneously producing more interferon (IFN)-γ than IBS controls. About 30% of IFN-γ-producing CD45(+) cells were T-bet(+), CD56(-), NKP44(-), and CD117(-), defining them as a type-1 innate lymphoid cells (ILC1). IFN-γ-producing ILC1 cells significantly decreased in 10 patients analyzed 2 weeks after they resumed a wheat-free diet.

Conclusions: These data indicate that, in patients with active NCWS, IFN-γ-producing ILC1 cells infiltrate rectal mucosa and support a role for this innate lymphoid cell population in the pathogenesis of NCWS.

Conflict of interest statement

Guarantor of the article: Antonio Carroccio, MD.

Specific author contributions: A. Carroccio and F. Dieli had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: A. Carroccio, F. Dieli, D. Di Liberto, P. Mansueto, A. D’Alcamo. Acquisition of data: A. Carroccio, A. D’Alcamo, G. Iacono, P. Mansueto. Endoscopy study: G. Geraci. Cell and cytokine analyses: D. Di Liberto, M. Lo Pizzo, E. Lo Presti, F. Fayer, G. Guggino. Analysis and interpretation of data: A. Carroccio, F. Dieli, D. Di Liberto, M. Lo Pizzo, E. Lo Presti, F. Fayer, G. Guggino, A. D’Alcamo, P. Mansueto. Drafting of the manuscript: A. Carroccio, F. Dieli, D. Di Liberto. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: D. Di Liberto, E. Lo Presti.

Financial support: The study was supported by the Italian Foundation for Celiac Disease (FC) Grant for Project 013/2014.

Potential competing interests: None.

Figures

Figure 1
Figure 1
Leukocyte infiltration and cytokine expression in rectal biopsies of non-celiac wheat-sensitive (NCWS) patients on wheat challenge. (a) Mean percentages of leukocytes (live CD45+ cells) infiltrating the rectal tissue of NCWS patients on wheat challenge and irritable bowel syndrome (IBS) controls. (b) Percentage of CD45+ cells and (c) percentage of CD3− cells expressing different cytokines in the rectal mucosa of NCWS patients on wheat challenge and IBS controls. Data are expressed as mean±s.e.m. *P<0.05. (d) Representative dot plots showing the expression of several cytokines by CD3+ and CD3− subsets of CD45+ cells infiltrating the rectal mucosa of IBS controls (upper panel) and NCWS patients on wheat challenge (upper panel). IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; 7AAD, 7-aminoactinomycin D.
Figure 2
Figure 2
Type-1 innate lymphoid cells (ILC1) infiltrate the inflamed rectal mucosa of non-celiac wheat-sensitive (NCWS) patients upon wheat challenge. (a) Representative dot plots showing the gating strategy for the ex vivo identification and characterization of ILC1 in the rectal mucosa of NCWS patients. (b) Representative images of confocal analysis of T-bet (green), interferon (IFN)-γ (red), and negative CD3 (cyan) co-localization in the rectal mucosa of a wheat-challenged NCWS patient (upper panel) and of an irritable bowel syndrome (IBS) control (lower panel). The merge panels show triple staining of the analyzed ILC1. Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole). (c) IFN-γ production by ILC1 infiltrating the rectal mucosa of a NCWS patient and cultured for 24 h either alone (medium) or with Ionomycin/phorbol myristate acetate (PMA) or with a combination of interleukin (IL)-12, IL-15, and IL-18. 7AAD, 7-aminoactinomycin D.
Figure 3
Figure 3
Effect of wheat-free diet on type-1 innate lymphoid cells (ILC1) infiltrating the rectal mucosa. (a) Percentages of cytokine-producing CD45+ cells and CD3− cells infiltrating the rectal mucosa of non-celiac wheat-sensitive (NCWS) patients on wheat challenge and after wheat-free diet. Data are expressed as mean±s.e.m. *P<0.05; **P<0.01 (Mann–Whitney test). (b) Percentages of cytokine-producing CD3− cells infiltrating the rectal mucosa of five individual NCWS patients on wheat challenge and after wheat-free diet. (c) ILC1 before (or on wheat challenge) and after wheat-free diet in NCWS patients. Data are shown as mean percentage±s.e.m. *P<0.05. (d) Representative dot plots showing the percentage of ILC1 in irritable bowel syndrome (IBS) control (upper panel) and NCWS patients on wheat challenge (middle panel) and after wheat-free diet (lower panel). FSC, forward scatter; IFN, interferon; SSC, side scatter; 7AAD, 7-aminoactinomycin D.

References

    1. Sapone A, Bai JC, Ciacci C et al. Spectrum of gluten related disorders: consensus on new nomenclature and classification. BMC Med 2012; 10: 13.
    1. Catassi C, Bai JC, Bonaz B et al. Non-celiac gluten sensitivity: the new frontier of gluten related disorders. Nutrients 2013; 5: 3839–3853.
    1. Catassi C, Elli L, Bonaz B et al. Diagnosis of non-celiac gluten sensitivity (NCGS): the Salerno Experts’ Criteria. Nutrients 2015; 7: 4966–4977.
    1. Carroccio A, Rini G, Mansueto P. Non-celiac wheat sensitivity is a more appropriate label than non-celiac gluten sensitivity. Gastroenterology 2014; 146: 320–321.
    1. Fasano A, Sapone A, Zevallos V et al. Non-celiac gluten-sensitivity. Gastroenterology 2015; 148: 1195–1204.
    1. Biesiekierski JR, Peters SL, Newnham ED et al. No effects of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates. Gastroenterology 2013; 145: 320–328.
    1. Peters SL, Biesiekierski JR, Yelland GW et al. Randomised clinical trial: gluten may cause depression in subjects with non-coeliac gluten sensitivity: an exploratory randomised clinical study. Aliment Pharmacol Ther 2014; 39: 1104–1112.
    1. Sapone A, Lammers KM, Mazzarella G et al. Differential mucosal IL-17 expression in two gliadin-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease. Int Arch Allergy Immunol 2010; 152: 75–80.
    1. Sapone A, Lammers KM, Casolaro V et al. Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity. BMC Med 2011; 9: 23.
    1. Brottveit M, Beitnes AC, Tollefsen S et al. Mucosal cytokine response after short-term gluten challenge in celiac disease and non-celiac gluten sensitivity. Am J Gastroenterol 2013; 108: 842–850.
    1. Vazquez-Roque MI, Camilleri M, Smyrk T et al. A controlled trial of gluten-free diet in patients with irritable bowel syndrome-diarrhea: effects on bowel frequency and intestinal function. Gastroenterology 2013; 144: 903–911.
    1. Cherrier M, Sawa S, Eberl G. Notch, Id2 and RORγt, sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J Exp Med 2012; 209: 729–740.
    1. Hoyler T, Klose CSN, Souabni A et al. Gata3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity 2012; 37: 634–648.
    1. Spits H, Cupedo T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function. Annu Rev Immunol 2012; 30: 647–675.
    1. Spits H, Di Santo JP. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nat Immunol 2011; 12: 21–27.
    1. Veldhoen M, Withers DR. Immunology. Innate lymphoid cell relations. Science 2010; 330: 594–595.
    1. Biron CA, Nguyen KB, Pien GC et al. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 1999; 17: 189–220.
    1. Di Santo JP. Natural killer cells: diversity in search of a niche. Nat Immunol 2008; 9: 473–475.
    1. Kiessling R, Klein E, Pross H et al. “Natural” killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell. Eur J Immunol 1975; 5: 117–121.
    1. Orange JS, Ballas ZK. Natural killer cells in human health and disease. Clin Immunol 2006; 118: 1–10.
    1. Yokoyama WM, Kim S, French AR. The dynamic life of natural killer cells. Annu Rev Immunol 2004; 22: 405–429.
    1. Ganal SC, Sanos SL, Kallfass C et al. Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota. Immunity 2012; 37: 171–186.
    1. Schulthess J, Meresse B, Ramiro-Puig E et al. Interleukin-15-dependent NKp46(+) innate lymphoid cells control intestinal inflammation by recruiting inflammatory monocytes. Immunity 2012; 37: 108–121.
    1. Wong SH, Walker JA, Jolin HE et al. Transcription factor RORalpha is critical for nuocyte development. Nat Immunol 2012; 13: 229–236.
    1. Sonnenberg GF, Fouser LA, Artis D. Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22. Nat Immunol 2011. a; 12: 383–390.
    1. Sonnenberg GF, Monticelli LA, Elloso MM et al. CD4(+) lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 2011. b; 34: 122–134.
    1. Geremia A, Arancibia-Cárcamo CV, Fleming MP et al. IL-23-responsive innate lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011; 208: 1127.
    1. Takayama T, Kamada N, Chinen H et al. Imbalance of NKp44(+)NKp46(-) and NKp44(-)NKp46(+) natural killer cells in the intestinal mucosa of patients with Crohn’s disease. Gastroenterology 2010; 139: 882.
    1. Bernink JH, Peters CP, Munneke M et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol 2013; 14: 221.
    1. Fuchs A, Vermi W, Lee JS et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-γ-producing cells. Immunity 2013; 38: 769.
    1. Carroccio A, Mansueto P, Iacono G et al. Non-celiac wheat sensitivity diagnosed by double-blind placebo-controlled challenge: exploring a new clinical entity. Am J Gastroenterol 2012; 107: 1898–1906.
    1. Carrasco A, Mañe J, Santaolalla R et al. Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa. J Immunol Methods 2013; 389 (1-2): 29–37.
    1. Carroccio A, D'Alcamo A, Cavataio F et al. High proportions of people with non-celiac wheat sensitivity have autoimmune disease or anti-nuclear antibodies. Gastroenterology 2015; 149: 596–603.
    1. Carroccio A, Mansueto P, D'Alcamo A et al. Non-celiac wheat sensitivity as an allergic condition: personal experience and narrative review. Am J Gastroenterol 2013; 108: 1845–1852.
    1. Nijeboer P, Bontkes HJ, Mulder CJ et al. Non-celiac gluten sensitivity. Is it in the gluten or the grain? J Gastrointestin Liver Dis 2013; 22: 435–440.
    1. Mooney PD, Aziz I, Sanders DS. Non-celiac gluten sensitivity: clinical relevance and recommendations for future research. Neurogastroenterol Motil 2013; 25: 864–871.
    1. Guandalini S, Polanco I. Non-celiac gluten sensitivity or wheat intolerance syndrome? J Pediatr 2015; 166: 805–811.
    1. Volta U, Caio G, Tovoli F et al. Non-celiac gluten sensitivity: questions still to be answered despite increasing awareness. Cell Mol Immunol 2013; 10: 383–392.
    1. Hollon J, Puppa EL, Greenwald B et al. Effect of gliadin on permeability of intestinal biopsy explants from celiac disease patients and patients with non-celiac gluten sensitivity. Nutrients 2015; 7: 1565–1576.
    1. Junker Y, Zeissig S, Kim S et al. Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of Toll-like receptor 4. J Exp Med 2012; 209: 2395–2408.
    1. Buonocore S, Ahern PP, Uhlig HH et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 2010; 464: 1371–1375.
    1. Ohman L, Simrén M. Pathogenesis of IBS: role of inflammation, immunity and neuroimmune interactions. Nat Rev Gastroenterol Hepatol 2010; 7: 163–173.
    1. Camilleri M, Lasch K, Zhou W. Irritable bowel syndrome: methods, mechanisms, and pathophysiology. The confluence of increased permeability, inflammation, and pain in irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol 2012; 303: G775–G785.
    1. Mansueto P, D’Alcamo A, Seidita A et al. Food allergy in irritable bowel syndrome: the case of non-celiac wheat sensitivity. World J Gastroenterol 2015; 21: 7089–7109.

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