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. 2017 Oct 1;11(10):1247-1257.
doi: 10.1093/ecco-jcc/jjx075.

Genetic Mouse Models with Intestinal-Specific Tight Junction Deletion Resemble an Ulcerative Colitis Phenotype

Wolfgang Stremmel  1 , Simone Staffer  1 , Mathias Jochen Schneider  2 , Hongying Gan-Schreier  1 , Andreas Wannhoff  1 , Nicole Stuhrmann  1 , Annika Gauss  1 , Hartwig Wolburg  3 , Anne Mahringer  4 , Alexander Swidsinski  5 , Thomas Efferth  2
Affiliations

Genetic Mouse Models with Intestinal-Specific Tight Junction Deletion Resemble an Ulcerative Colitis Phenotype

Wolfgang Stremmel et al. J Crohns Colitis. .

Abstract

Background and aims: A key pathogenetic feature of ulcerative colitis [UC] is an intrinsic low mucus phosphatidylcholine[PC] content. Recently, a paracellular transport for PC across tight junctions[TJs] was described, suggesting TJ disturbance as a cause of diminished luminal PC transport. Therefore, we aimed to generate mutant mice with TJ deletion to evaluate whether a UC phenotype developed.

Methods: CL57BL/6 control wild-type mice were compared to mutant mice with tamoxifen-induced villin-Cre-dependent intestinal deletion of kindlin 1 and 2.

Results: Electron microscopy of mucosal biopsies obtained from both mutants before overt inflammation following only 2 days of tamoxifen exposure revealed a defective TJ morphology with extended paracellular space and, by light microscopy, expanded mucosal crypt lumina. PC secretion into mucus was reduced by >65% and the mucus PC content dropped by >50%, causing a >50 % decrease of mucus hydrophobicity in both mutants. Consequently, the microbiota was able to penetrate the submucosa. After 3 days of tamoxifen exposure, intestinal inflammation was present in both mutants, with loose bloody stools as well as macroscopic and histological features of colitis. Oral PC supplementation was able to suppress inflammation. By analogy, colonic biopsies obtained from patients with UC in remission also showed a defective epithelium with widened intercellular clefts, and enlarged crypt luminal diameters with functionally impaired luminal PC secretion.

Conclusions: Genetic mouse models with intestinal deletion of kindlin 1 and 2 resulted in TJ deletion and revealed pathophysiological features of impaired PC secretion to the mucus leading to mucosal inflammation compatible with human UC.

Keywords: Mucosal barrier; hydrophobicity; mucus; phosphatidylcholine; ulcerative colitis.

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Figures

Figure 1.
Figure 1.
Pre-symptomatic features of ileal mucosa in the genetic mouse models of UC versus wild-type controls. [A] Electron micrographs of freeze-fracture replicas showing a close network of TJs in controls and TJ disturbance with open ends of the strands in the mutants [arrows mark open ends of TJ strands; scale bars = 0.2 µm]. [B] Widening of crypt diameters in mutants [HE staining]. [C] NBD-PC life exposure of ileal biopsies showing in controls a cylindrical, tightly packed arrangement of enterocytes with paracellular and mucus fluorescent staining. In mutants the cells are cuboidal with extended paracellular spaces and impaired PC staining, in particular of the mucus [cuboidal cells in kindlin 1(−/−) and kindlin 2(−/−) mice are indicated by white arrows]. [D] Reduced luminal Pearse and PAS phospholipid staining in mutants compared to controls [scale bars in B–D = 25 µm]. [E] FISH [1000-fold magnification] of segmented filamentous bacteria penetrating the ileal mucosa in mutants but not in controls.
Figure 2.
Figure 2.
Crypt diameters and intestinal PC secretion capacity versus inulin and PI in control and mutant mice after 2 days of tamoxifen exposure [pre-inflammatroy state]. [A] Ratio of luminal diameter [dgap] to total crypt diameter [dcrypt]. Colon: control, 0.084 ± 0.016; kindlin 1(−/−), 0.155 ± 0.028; kindlin 2(−/−), 0.223 ± 0.019. Ileum: control, 0.097 ± 0.016; kindlin 1(−/−), 0.231 ± 0.040; kindlin 2(−/−), 0.184 ± 0.049 [P < 0.01 mutants vs. controls] [n = 6]. [B] In vivo-determined secretion rates at 16 h after intravenous administration of substrates revealed suppression of mucus recovery of [3H]PC in ileum and colon of kindlin 1(−/−) and 2(−/−) mice. The recovery rates of [14C]inulin and [3H]PI were unaltered [n = 12]. Means ± SD. ***P < 0.01; ns: not significant [mutants vs. controls].
Figure 3.
Figure 3.
Western blot of isolated mucosal cells from control and mutant mice. Blots were stained with antibodies to proteins of the kindlin to β1-integrin activation to E-cadherin [adherence junction] and to representative TJ proteins [ZO1, occludin, claudin2].
Figure 4.
Figure 4.
Macroscopic, stool, ileal endoscopic and histological features of wild-type control mice and kindlin 1(−/−) and 2(−/−) mice, without or with oral PC feeding. Five-centimeter sections of the terminal ileum were dissected, while the colon was entirely removed. Top: magnified sections of the closed and opened gut segments and stool samples. Bottom: endoscopic and histological features of the mutant mice, without and with PC pretreatment [scale bars = 25 μm].
Figure 5.
Figure 5.
Widened crypts due to disturbed TJs in human UC [in remission] and consequent impairment of luminal PC accumulation. [A] Electron micrograph of a human UC specimen with epithelial disturbance [arrow shows widening of the intercellular cleft] and HE staining of non-inflamed mucosa with wider crypt lumina in UC patients than in control subjects and patients with Crohn’s disease. [B] [upper panel] NBD-PC live exposure of colonic biopsies showing an impaired paracellular and mucus staining only in UC patients but not in healthy controls and patients with Crohn’s disease. [lower panels] Reduced Pearse and PAS phospholipid staining of samples from UC patients in clinical remission vs. control subjects and patients with Crohn’s disease [scale bars = 25 µm].
Figure 6.
Figure 6.
Scheme illustrating the proposed pathophysiological events in UC. Phosphatidylcholine [PC] in mucus establishes a hydrophobic shield against colonic microbiota. Originating from plasma lipoproteins and the segregated lipoprotein-free fraction, PC selectively accumulates in mucus via paracellular, tight junction [TJ]-dependent translocation. Transport is driven by a negative electrical gradient, with consequent binding to membrane-localized mucin 3 and an equilibrated shift to secretory mucin 2. In UC, disturbance of the TJ prevents paracellular PC secretion, resulting in a low mucus PC content, and thus reduced hydrophobicity. This predisposes the colon to microbiota invasion and mucosal inflammation.
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