Functional intraepithelial lymphocyte changes in inflammatory bowel disease and spondyloarthritis have disease specific correlations with intestinal microbiota

Abstract

Background: Dysbiosis occurs in spondyloarthritis (SpA) and inflammatory bowel disease (IBD), which is subdivided into Crohn's disease (CD) and ulcerative colitis (UC). The immunologic consequences of alterations in microbiota, however, have not been defined. Intraepithelial lymphocytes (IELs) are T cells within the intestinal epithelium that are in close contact with bacteria and are likely to be modulated by changes in microbiota. We examined differences in human gut-associated bacteria and tested correlation with functional changes in IELs in patients with axial SpA (axSpA), CD, or UC, and in controls.

Methods: We conducted a case-control study to evaluate IELs from pinch biopsies of grossly normal colonic tissue from subjects with biopsy-proven CD or UC, axSpA fulfilling Assessment of SpondyloArthritis International Society (ASAS) criteria and from controls during endoscopy. IELs were harvested and characterized by flow cytometry for cell surface markers. Secreted cytokines were measured by ELISA. Microbiome analysis was by 16S rRNA gene sequencing from rectal swabs. Statistical analyses were performed with the Kruskal-Wallis and Spearman's rank tests.

Results: The total number of IELs was significantly decreased in subjects with axSpA compared to those with IBD and controls, likely due to a decrease in TCRβ+ IELs. We found strong, significant negative correlation between peripheral lymphocyte count and IEL number. IELs secreted significantly increased IL-1β in patients with UC, significantly increased IL-17A and IFN-γ in patients with CD, and significantly increased TNF-α in patients with CD and axSpA as compared to other cohorts. For each disease subtype, IELs and IEL-produced cytokines were positively and negatively correlated with the relative abundance of multiple bacterial taxa.

Conclusions: Our data indicate differences in IEL function among subjects with axSpA, CD, and UC compared to healthy controls. We propose that the observed correlation between altered microbiota and IEL function in these populations are relevant to the pathogenesis of axSpA and IBD, and discuss possible mechanisms.

Trial registration: ClinicalTrials.gov, NCT02389075 . Registered on 17 March 2015.

Keywords: Crohn’s disease; Inflammatory bowel disease; Intraepithelial lymphocytes; Microbiome; Spondyloarthritis; Ulcerative colitis.

Conflict of interest statement

Ethics approval and consent to participate

All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The study protocol was approved by the Colorado Multiple Institutional Review Board. All patients provided written informed consent and authorization for release of personal health information. An independent safety officer was assigned and met every 12 months with the investigators to conduct safety reviews.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Colonic intraepithelial lymphocytes (IELs) are significantly decreased in individuals with axial spondyloarthritis (axSpA) and inversely correlate with peripheral blood lymphocyte counts. IELs were obtained from colonic mucosal biopsies as described in “Methods” and evaluated by flow cytometry for the absolute number of total IELs (a), T cell receptor (TCR)β+ IELs (b), and TCRγδ+ IELs (c). Each dot represents an individual within the study group identified on the x-axis. A solid square indicates subjects on a TNF inhibitor, a solid dot indicates the subject was taking steroids, and an open dot indicates the subject was taking neither. Bars are the mean ± SEM. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-hoc analysis. d Total IELs (y-axis) in each individual with axSpA were compared to the absolute lymphocyte count (x-axis) and the Pearson’s correlation coefficient was calculated (σ = − 0.94, R2 = 0.89, p = 0.0047). HC, healthy controls; CD, Crohn’s disease; UC, ulcerative colitis

Fig. 2

Cytokines produced by intraepithelial lymphocytes (IELs) are altered in individuals with inflammatory bowel disease (IBD) and axial spondyloarthritis (axSpA). Colonic IELs were mitogen-stimulated overnight and secreted cytokines measured by ELISA. a IL-1β, b IL-17A, c interferon (IFN)-γ, and d TNF-α production in pg/ml was normalized to the number of IELs collected from each subject. Each dot represents a case/control analyzed. A solid square indicates subjects on a TNF inhibitor, a solid dot indicates the subject was taking steroids, and an open dot indicates the subject was taking neither. Subjects with undetectable cytokine levels are absent from the graphs due to the logarithmic scale as are means that fall below the axis range. Statistical differences were identified by the Kruskal-Wallis test with Dunn’s post-hoc analysis for pairwise differences: *p < 0.05 and **p < 0.01. HC, healthy controls; CD, Crohn’s disease; UC, ulcerative colitis; axSpA, axial spondyloarthritis

Fig. 3

Microbial community comparisons in individuals with inflammatory bowel disease (IBD) and axial spondyloarthritis (axSpA). Bacterial DNA from fecal swabs from patients (cases) and controls were sequenced for 16S rRNA and analyzed. a The percent abundance of the top operational taxonomic unit (OTU) families were compared across subject groups. Differences in the overall composition of microbial communities were determined by permutation-based multiple analysis of variance. b–d Pairwise comparisons of OTUs between disease states and controls were performed using the Wilcoxon rank-sum test. The mean relative abundance ± SEM for each OTU that was statistically significant (p < 0.05) is shown for Crohn’s disease (CD) (b), ulcerative colitis (UC) (c), and axSpA (d). HC, healthy controls

Fig. 4

There was disease-specific correlation between microbiota and intraepithelial lymphocyte (IEL) production of cytokines. Correlation between the operational taxonomic unit (out) relative abundance and the IEL-produced cytokine value in each case/control group was tested using Spearman’s rank test. The data are shown as heatmaps, with the color of each correlation test corresponding to the Spearman rho value: *p < 0.05, †p < 0.01, and ‡p < 0.001. HC, healthy controls; CD, Crohn’s disease; UC, ulcerative colitis; axSpA, axial spondyloarthritis