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Comparative Study
. 2005 Dec 13;102(50):18129-34.
doi: 10.1073/pnas.0505256102. Epub 2005 Dec 5.

Reduced Paneth cell alpha-defensins in ileal Crohn's disease

Jan Wehkamp  1 , Nita H Salzman, Edith Porter, Sabine Nuding, Michael Weichenthal, Robert E Petras, Bo Shen, Elke Schaeffeler, Matthias Schwab, Rose Linzmeier, Ryan W Feathers, Hiutung Chu, Heriberto Lima Jr, Klaus Fellermann, Tomas Ganz, Eduard F Stange, Charles L Bevins
Affiliations
Comparative Study

Reduced Paneth cell alpha-defensins in ileal Crohn's disease

Jan Wehkamp et al. Proc Natl Acad Sci U S A. .

Abstract

The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.

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Figures

Fig. 1.
Fig. 1.
PC α-defensins in controls and IBD patients. (A) Illustration of the position of PCs at the base of the crypt of Lieberkühn in the small intestinal mucosa (illustration by David R. Schumick Illustration, Elyria, OH). (B) Phloxine tartrazine staining of small intestinal ileal mucosa (Left), showing antimicrobial peptide-rich granules. Immunohistochemical localization of the PC α-defensin HD5 (Right). (Upper) Normal control; (Lower) CD. (Scale bars, 25 μm.) (C) Expression of HD5 mRNA in surgical specimens from controls and patients with ileal CD. The mRNA copy number per 10 ng of total RNA was determined with quantitative real-time RT-PCR using external standards. [Scale bars represent means (±standard error).] The significance values are based on the Mann–Whitney test (*, P < 0.05). (D) Expression of HD5 in patients with ileal CD with respect to NOD2/CARD15 genotype. Note that compared with non-IBD controls, all NOD2/CARD15 genotype subgroups (wild type and mutated) of ileal CD presented here have significantly reduced HD5 mRNA levels, as shown in C. Data expressed and analyzed as in C. (E) (Upper) Coomassie blue-stained SDS gel containing protein extracts (12 μg per lane) from ileal mucosa of controls (lanes 1–3) and ileal CD patients (lanes 4–9). NOD2/CARD15 genotype analysis detected SNP-13 mutation in ileal CD patients (lanes 7–9); wild-type sequence was found in other samples (lanes 1–6). Standards are recombinant proHD5 (open arrow). (Lower) Immunoblot analysis of HD5 peptide in ileal tissue samples. Blot using HD5 antibody (36, 39) was from replicate gel as in A with 0.6 μg per lane protein loading. The difference in mean values between controls (lanes 1–3) and ileal CD specimens (lanes 4–9) was significant by t test analysis (P = 0.038). (F) Quantification of HD5 peptide in ileal tissue samples. Scale bars represent the percentage (±standard error) of HD5 peptide amounts in CD specimens as compared with nondisease control samples, which was set as 100%. HD5 peptide concentrations in ileal samples were determined by quantitative comparison of immunoblot bands from tissue to serial dilutions of recombinant HD5 peptide on the same gel/membrane (data not shown). Signal was quantified by using a VersaDoc 1000 BioRad imaging system. (G) Immunoblot blot analysis of sPLA2, lysozyme, and α-1-antiprotease. (Upper) Coomassie blue staining of an SDS-Tricine PAGE gel (12 μg/lane), as described in E.(Lower) Analysis from replicate gel as in E with 1.2 μg per lane protein loading with sPLA2, lysozyme, and α-1-antiprotease antibodies.
Fig. 2.
Fig. 2.
Mucosal inflammation and expression of PC defensin HD5. (A) Correlation of mucosal inflammation and the proinflammatory cytokine IL-8 mRNA levels in ileal CD and non-IBD controls. Hematoxylin/eosin-stained paraffin sections from specimens were assessed for mucosal inflammation by a gastrointestinal pathologist who was uninformed about samples [no inflammation (0), moderate (1), and severe (2)]. IL-8 mRNA expression levels, expressed as mRNA copies per 10 ng of RNA [means (±standard error) shown for each group]. Inset shows the average inflammation score for controls (n = 8) and ileal CD patients (n = 25). (B) Expression of HD5 mRNA in specimens from non-IBD controls and patients with ileal CD grouped as in A. Data expressed as mRNA copy number per 10 ng RNA (means ± standard error), from same specimens as analyzed in Fig. 1C. (C) Expression of IL-8 mRNA in specimens from normal and inflamed ileal pouches. Biopsy specimens were from normal (n = 7) and inflamed pouch mucosa (pouchitis, n = 7). IL-8 mRNA expression levels are expressed as in A. The significance values based on the Mann–Whitney test (*, P < 0.05). (D) Expression of HD5 mRNA in the same specimens as in C. A similar pattern was observed for HD6 (data not shown).
Fig. 3.
Fig. 3.
Quantitative analysis of antimicrobial activity and PC mRNA transcript copy numbers in ileal mucosa. (A) Antimicrobial activity in mucosal biopsies of ileal CD and non-IBD controls. Protein extracts were incubated with cultures of either E. coli (Left)or S. aureus (Right), and bacterial killing was assessed by using a flow cytometric assay. Biopsies from ileal CD (all NOD2 wild type) were obtained from either macroscopically inflamed (open circles) or uninflamed (filled diamonds) regions of the ileum. (B) Expression levels of four PC antimicrobials (on the left) and six nonantimicrobial PC products in ileal specimens of controls and ileal CD. Data are compiled from experiments described in Figs. 1 and 5 and are expressed as mRNA copy number per 10 ng of RNA.
Fig. 4.
Fig. 4.
Bacterial microbiota in wild-type, heterozygous, and homozygous HD5 TG mice. (A) Expression and localization of HD5 mRNA in TG mouse small intestine analyzed by in situ hybridization by using an antisense probe (Left). Hybridization of HD5 probe to section of human ileum (Inset). Wild-type mouse shows no hybridization to HD5 antisense probe (data not shown). Sense probe (Right) and RNase A pretreatment controls (data not shown) were negative for hybridization signal. Arrows point to dense signals that overlie PCs. Counterstain was hematoxylin/eosin. (Scale bar, 20 μm.) (B) Expression of HD5 mRNA in ileal specimens from heterozygous and homozygous HD5 TG mice. Data expressed as mRNA copy number per 10 ng of total RNA determined with quantitative real-time RT-PCR using external standards. [Scale bars represent means (±standard error).] (C) FISH analysis of luminal microbes in mouse ileum. Representative hybridization analysis with TR-Bac338 probe (detecting all bacteria) is shown for wild-type mice (Left), HD5 TG heterozygote mice (Center) and HD5 TG homozygote mice (Right).
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