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. 2014 Jan;146(1):200-9.
doi: 10.1053/j.gastro.2013.09.048. Epub 2013 Sep 25.

Genetic variants synthesize to produce paneth cell phenotypes that define subtypes of Crohn's disease

Kelli L VanDussen  1 , Ta-Chiang Liu  1 , Dalin Li  2 , Fadi Towfic  3 , Nir Modiano  4 , Rachel Winter  4 , Talin Haritunians  2 , Kent D Taylor  5 , Deepti Dhall  6 , Stephan R Targan  4 , Ramnik J Xavier  3 , Dermot P B McGovern  7 , Thaddeus S Stappenbeck  8
Affiliations

Genetic variants synthesize to produce paneth cell phenotypes that define subtypes of Crohn's disease

Kelli L VanDussen et al. Gastroenterology. 2014 Jan.

Abstract

Background & aims: Genetic susceptibility loci for Crohn's disease (CD) are numerous, complex, and likely interact with undefined components of the environment. It has been a challenge to link the effects of particular loci to phenotypes of cells associated with pathogenesis of CD, such as Paneth cells. We investigated whether specific phenotypes of Paneth cells associated with particular genetic susceptibility loci can be used to define specific subtypes of CD.

Methods: We performed a retrospective analysis of 119 resection specimens collected from patients with CD at 2 separate medical centers. Paneth cell phenotypes were classified as normal or abnormal (with disordered, diminished, diffuse, or excluded granule phenotypes) based on lysozyme-positive secretory granule morphology. To uncover the molecular basis of the Paneth cell phenotypes, we developed methods to determine transcriptional profiles from whole-thickness and laser-capture microdissected, formalin-fixed, paraffin-embedded tissue sections.

Results: The proportion of abnormal Paneth cells was associated with the number of CD-associated NOD2 risk alleles. The cumulative number of NOD2 and ATG16L1 risk alleles had an additive effect on the proportion of abnormal Paneth cells. Unsupervised clustering analysis of demographic and Paneth cell data divided patients into 2 principal subgroups, defined by high and low proportions of abnormal Paneth cells. The disordered and diffuse abnormal Paneth cell phenotypes were associated with an altered transcriptional signature of immune system activation. We observed an inverse correlation between abnormal Paneth cells and presence of granuloma. In addition, high proportions of abnormal Paneth cells were associated with shorter time to disease recurrence after surgery.

Conclusions: Histologic analysis of Paneth cell phenotypes can be used to divide patients with CD into subgroups with distinct pathognomonic and clinical features.

Keywords: CD; Crohn's disease; Diagnosis; IBD; Inflammatory Bowel Disease; Pathogenesis; Prognostic Factor; UC; inflammatory bowel disease; ulcerative colitis.

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Figures

Figure 1
Figure 1. CD-associated NOD2 risk alleles are associated with abnormal Paneth cell morphology
(A–C) Lysozyme immunostaining (red) was performed to visualize and score Paneth cell secretory granule morphology from cases with 0 (n = 29), 1 (n = 25) or 2 (n = 5) CD-associated NOD2 risk variants. Paneth cells were scored as normal if they contained numerous small (~1 μm), lysozyme-positive apically located granules. Disordered Paneth cells contained lysozyme-positive granules of normal size and quantity, but had some basally located granules. Diminished Paneth cells contained <10 granules, with the remaining granules frequently enlarged or fused. Diffuse Paneth cells did not contain any secretory granules and had diffuse lysozyme staining throughout their cytoplasm. (A) Representative images of lysozyme immunostaining. Nuclear counterstain, DAPI (blue). Well-oriented Paneth cells are outlined in white and the scored phenotype is indicated: normal (N), disordered (Dis), diminished (Dim), diffuse (Dif) or excluded granule (Exc). Bars = 10 μm. (B) Representative images of Paneth cells with the excluded granule phenotype (arrowheads). (C) Quantification of Paneth cell phenotypes according to the number of CD-associated NOD2 risk variants. (D) Quantification of the percent of abnormal Paneth cells in cases with 0 CD-associated NOD2 susceptibility alleles (n = 29) or in cases that carried one allele of R702W (n = 10), G908R (n = 4) or L1007fsXinsC (n = 9). Data are presented as the mean ± s.e.m. of the percent of Paneth cells with the indicated phenotype out of the total number of Paneth cells counted for each case. Cases with the ATG16L1 T300A variant were excluded from these analyses. Permutation tests were performed to demonstrate the statistically significant association between the presence of CD-associated NOD2 variants and the percentage of abnormal Paneth cells for each scoring category (*, P < 0.05; **, P < 0.01). Mann-Whitney tests were used to demonstrate statistical difference between the presence of 1 or 2 NOD2 risk variants and controls (†, P < 0.05; ††, P < 0.01).
Figure 2
Figure 2. Abnormal Paneth cell phenotypes define a distinct subtype of CD cases
(A) Total abnormal Paneth cell proportions were examined in a larger cohort (n = 119) with cumulative number of ATG16L1 T300A and CD-associated NOD2 variants (including common and rare variants) 0 (n = 29), 1 (n = 41), 2 (n = 33), 3 (n = 10) or 4 (n = 6). Linear regression analysis demonstrated a significant, positive correlation between the percent of abnormal Paneth cells per case and cumulative risk alleles. (B) Unsupervised clustering was performed for the CD cases with Paneth cell phenotype, genetic, documented environmental exposure and demographic information available. Cases are arranged in the same order along the x and y axes. The Pearson correlation was determined for each patient-patient comparison, and then patients were clustered according to similarity with the results represented as a heat map. (C) Heat map displaying the factors that significantly contribute to the identification of Subtypes 1 and 2 in (B), which were defined by low and high proportions of abnormal Paneth cells, respectively. Heat map cells are colored red and blue to indicate positive and negative correlations, respectively. PCs, Paneth cells.
Figure 3
Figure 3. A specific gene signature associated with an activated immune response correlates with the disordered and diffuse Paneth cell phenotypes
The 114 and 218 transcripts with expression values that correlated with the disordered and diffuse Paneth cell phenotype, respectively, were analyzed using DAVID to identify biological process gene ontology (GO) terms significantly overrepresented in each gene set. (A, B) GO terms related to the immune response are indicated (fully labeled bar graphs are shown in Figures S8 and S9). Bars are colored according to the major GO term category: biological process (gray), cellular component (white), molecular function (black). PC, Paneth cell.
Figure 4
Figure 4. Abnormal Paneth cell phenotypes and presence of granuloma are inversely correlated
The incidence of granuloma was determined in our expanded CD cohort (n = 107). Correlation of granuloma incidence and cases with low (white bars) vs. high proportions (black bars) of (A) abnormal Paneth cell phenotypes (cutoff ≥ 20%; n = 63 [< 20%], n = 36 [≥ 20%]), (B) ‘excluded granule’ Paneth cell phenotypes (cutoff ≥ 5%; n = 71 [< 1%], n = 28 [≥1%]), (C) diminished granules (cutoff ≥ 10%; n = 42 [< 10%], n = 57 [≥ 10%]) and (D) diffuse granules (cutoff ≥ 5%; n = 92 [< 5%], n = 7 [≥ 5%]). Statistical significance was determined by Chi-Square test (*P ≤ 0.05).
Figure 5
Figure 5. Abnormal Paneth cell phenotypes correlate with shorter time to disease recurrence in CD patients that received prophylaxis post-surgery
The time to disease recurrence post-resection surgery was determined in the CD cases that received prophylaxis post-surgery (n = 102). Prophylaxis included use of immunomodulators (e.g., 6-mercaptopurine, azathioprine, methotrexate) or biologics (i.e., anti-TNF therapy). Cases were assigned to the “Abnormal Paneth cells: Low” or “Abnormal Paneth cells: High” groups using a cutoff of 20% abnormal Paneth cell phenotypes. Data are presented as a survival curve with *P = 0.0200 by Log-rank test.
Figure 6
Figure 6. Proposed model for the use of disease-relevant phenotypes as integrative readouts for the stratification of CD patients
Abnormal Paneth cell phenotypes can synthesize multiple genetic and environmental inputs. These phenotypes are associated with molecular and pathological features and can be used to subdivide CD patients.
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