Loss of Sonic hedgehog leads to alterations in intestinal secretory cell maturation and autophagy.

BACKGROUND: Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. METHODOLOGY/PRINCIPAL FINDINGS: A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. CONCLUSIONS/SIGNIFICANCE: Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy

Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes.

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella

Loss of intestinal epithelial Shh signaling deregulates intestinal crypt epithelial proliferation.

<p>Hematoxylin and eosin staining was performed on ileal paraffin sections of 180-day-old control (A) or <i>Shh</i>^ΔIEC (B) mice. The length of the crypt/villus axis was determined using MetaMorph v7.7 software and statistical analysis revealed a 1.2-fold reduction in crypt-villus axis length in <i>Shh</i>^ΔIEC ileum when compared to controls (C) (n = 4). Apoptosis assays by TUNEL staining were performed on paraffin sections of 180-day-old control (D) or <i>Shh</i>^ΔIEC (E) mice. DAPI (blue staining) served as a counterstain. No modulation in the number of TUNEL-positive cells was observed between mutant and controls (F). Proliferation assays were performed by PCNA immunostaining (G and H, green labeling), with Evans blue serving as counterstain (red staining). Mutants displayed a decrease in cell proliferation as shown by a decrease in the number of PCNA-labeled proliferating cells (H) when compared to controls (G). The number of PCNA-positive cells was quantified in the ileum of both controls and mutants (n = 4). Statistical analysis of the number of positive PCNA cells revealed a significant 1.3-fold decrease in proliferation in mutant animals (I). (n = 4) Two-way ANOVA ***<i>p</i><0.001. Scale bar: 50 µm. Error bars represent SEM.</p

Loss of intestinal epithelial Shh signaling does not activate the Wnt/β-catenin pathway.

<p>Immunostaining with an anti-β-catenin antibody revealed no modulation of nuclear translocation of β-catenin (black arrows) between controls (A) and mutant mice (B). Immunostaining with an anti-E-cadherin antibody in both control (C) and mutant mice (D) enabled to determine the localization of the cell membrane relative to that of the nucleus. Western blot analysis for c-Myc and cyclin D1/D2 classical targets of epithelial cell proliferation was performed on ileal extracts from control and <i>Shh</i>^ΔIEC mice (E). Densitometry analysis of exposed films using ImageJ revealed no significant modulation in c-Myc and cyclin D1/D2 expression levels in <i>Shh</i>^ΔIEC mice compared to controls (F) (n = 3) Student t-test. Scale bar: 50 µm.</p

Absorptive cell differentiation is not affected by the loss of intestinal epithelial Shh signaling.

<p>Immunostaining for intestinal fatty acid binding protein (iFABP) (green labeling) was performed on 180-day-old <i>Shh</i>^ΔIEC mice (B) and control littermates (A). Evans blue (red staining) served as a counterstain. Quantitative RT-PCR of sucrase-isomaltase revealed no modulation between mutant and control mice (C) (n = 7). Ultrastructural assessment of the apical membrane of ileal enterocytes from mutant mice (E) showed normal apical brush border and junctional complexes when compared to control littermates (D). Western blot analysis of claudin-1 and 2, Jam-A, occludin and E-cadherin was performed on total ileum lysates isolated from <i>Shh</i>^ΔIEC and control mice (F) (n = 4). Scale bar: 50 µm (A and B). Scale bar: 0.5 µm (D and E) Error bars represent SEM. SI, sucrase-isomaltase.</p

Enteroendocrine cells are not affected by the loss of intestinal epithelial Shh signaling.

<p>Chromogranin A was used as a general endocrine cell marker as it labels all subtypes of enteroendocrine cells. No modulation was observed in chromogranin A (CgA)-positive cells (green staining) in the ileal epithelium of control (A) and <i>Shh</i>^ΔIEC mice (B). Evans blue (red staining) served as a counterstain. The number of enteroendocrine (CgA)-positive cells was determined from the ileum of control and mutant mice (n = 4) (C). Scale bar: 50 µm. Two-way ANOVA. Error bars represent SEM.</p

Shh signaling is required for proper production of goblet cell secretory products and Paneth cell maturation.

<p>Alcian blue staining was performed to detect mucin (Muc2)-containing goblet cells in control (A) and mutant (B) littermates. Goblet cells were counted from ileum of control and <i>Shh</i>^ΔIEC mice (n = 4) with statistical analysis showing a significant 1.2-fold decrease in the number of acidic mucin-positive cells in mutant mice compared to controls (C). Immunostaining with an anti-lysozyme antibody showed a decrease in positively-labeled lysozyme cells (green staining) in mutant mice (E) compared to control littermates (D). Evans blue (red staining) served as a counterstain. Paneth cells were counted from ileum of control and <i>Shh</i>^ΔIEC mice (n = 4) and statistical analysis showed a significant 1.3 fold decrease in the number of lysozyme-positive cells in mutant mice compared to controls (F). Fucosylated residues in goblet and Paneth cells were analyzed using Ulex europeus-I agglutinin (UEA-I) lectin staining (G and H). UEA-1 staining exposed an important defect in fucosylation in ileal goblet cells in <i>Shh</i>^ΔIEC mice (H) when compared to controls (G), whereas fucosylation was not modulated in Paneth cells of <i>Shh</i>^ΔIEC mice (white arrows, H) when compared to controls (white arrowheads, G). Scale bar: 50 µm. Two-way ANOVA ***<i>p</i><0.001. Error bars represent SEM.</p

Loss of intestinal epithelial Shh signaling leads to an increase in ER stress and a decrease in autophagy in intestinal epithelial cells (IECs).

<p>Transmission electron microscopy revealed a dilated ER lumen in <i>Shh</i>^ΔIEC Paneth cells (black arrows, B) when compared to controls (black arrowheads, A). Ultrastructural examination revealed granule abnormalities in mutant mice (B). Paneth granules were counted in both <i>Shh</i>^ΔIEC and control mice and no significant modulation was found in the number of granules per Paneth cell between control and mutant mice (C) (n = 4). The mean size of Paneth cell granules was determined using MetaMorph software and statistical analysis revealed that granules were significantly smaller in <i>Shh</i>^ΔIEC ileum when compared to controls (D) (n = 4). Western blot analysis against defensin 4, IRE1α, p62, lipidated LC3b-II and non-lipidated LC3b-I proteins were performed on ileal extracts from control and <i>Shh</i>^ΔIEC mice (E). Densitometry analysis of exposed films using ImageJ revealed a significant 3.85-fold decrease in defensin 4 expression (F) a significant 3.6-fold increase in IRE1α (G) and a significant 2.13-fold increase in p62 (H) in <i>Shh</i>^ΔIEC mice compared to controls (n = 3). An 11-fold decrease in lipidated LC3b-II to non-lipidated LC3b-I ratio in <i>Shh</i>^ΔIEC mice can be seen compared to controls (I) (n = 3). Two-way ANOVA ***<i>p</i><0.001 (C–D). Student t-test *<i>p</i><0.05, **<i>p</i><0.01 (F–I). Error bar represents SEM. Scale bar: 2 µm. Def 4, defensin 4.</p