1. Volume 138, Issue 2, Pages 562-572.e2 (February 2010)
Interleukin-1β Promotes Gastric Atrophy Through Suppression of Sonic Hedgehog 
Meghna Waghray, Yana Zavros, Milena Saqui–Salces, Mohamad El–Zaatari, C. Bharath Alamelumangapuram, Andrea Todisco, Kathryn A. Eaton, Juanita L. Merchant 
Gastroenterology 
Volume 138, Issue 2, Pages e2 (February 2010)
DOI: /j.gastro
Copyright © 2010 AGA Institute Terms and Conditions

2. Figure 1
Shh is predominantly expressed in the corpus. Whole mounts of stomachs from a (A) nontransgenic and a (B) Shh–LacZ reporter mouse are shown after incubating the tissue in a β-galactosidase substrate (X-gal) for 16 hours. Quantitative reverse-transcription PCR was performed on corpus RNA from nontransgenic and Shh–LacZ reporter mice. (C) Shown is the ratio of Shh to GAPDH mRNA. The mean ± SEM for 3 mice is shown. (D) Immunoblots of protein isolated from the corpus of nontransgenic and Shh–LacZ mice are shown; the immunoblot for Shh was reblotted for GAPDH. Protein expression was quantified using image-J software (National Institutes of Health). (D) The mean ± SEM for Shh/GAPDH is shown. (E) Gastric tissue sections extending from the proximal portion of the stomach, the forestomach (black arrow), into the corpus is shown. Nuclear β-galactosidase activity was detected in the epithelial cells (X-gal and H&E staining). Ntg, nontransgenic.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
Both mucous and oxyntic cell lineages express Shh. Whole mounts of stomachs from Shh–LacZ reporter mice were incubated in the β-galactosidase substrate (X-gal) for 16 hours before paraffin embedding. The stomach was sectioned and then immunostained for cell-specific markers. (A) Ulex europaeus (uae1) for pit cells, (B) Griffonia simplicifolia (gsii) for mucous neck cells, (C) H+-K+-ATPase (hk) for parietal cells, and (D) intrinsic factor (if) for chief cells. Magnification, 400×; insets, 1000×.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
Helicobacter infection suppresses Shh expression. X-gal and H&E staining of 3-month-old (A) Shh–LacZ uninfected and (B) mice infected with H felis for 3 weeks and (C) 4-month-old uninfected and (D) mice infected with H felis for 8 weeks. Magnification, 200×; insets, 100×. (E and G) The number of H+,K+-ATPase–positive cells per gland at 3 and 8 weeks of infection is shown. Shh–LacZ–positive cells after (F) 3 weeks and (H) 8 weeks of H felis infection were analyzed by morphometry. Shown is the mean ± SEM for LacZ-positive cells from 6 mice per group for 3 weeks and 10 mice per group for 8 weeks *P < .05 relative to uninfected mice.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Loss of Shh expression precedes parietal cell atrophy. Immunohistochemical staining of parietal cells on X-gal–stained paraffin sections from (A) uninfected and (B) 8 weeks' H felis–infected mice. Magnification, 200×; inset, 1000×. (C) Morphometric analysis of the total number of parietal cells from uninfected and 8 weeks' H felis–infected mice. Changes in the parietal cells expressing Shh (X-gal) were analyzed for uninfected and 8 week–infected H felis mice. Shown is the mean ± SEM for 15 glands from 3 mice per group. *P < .05 relative to uninfected mice.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
IL-1β inhibits Shh expression. Quantitative reverse-transcription PCR was performed on total stomach RNA from uninfected and Shh–LacZ reporter mice infected with H felis for (A) 3 weeks (n = 6 uninfected, n = 4 infected), or (B) 8 weeks (n = 10). Shown is the relative induction of IL-1β mRNA normalized to GAPDH. The mean ± SEM is shown. *P < .05 compared with uninfected mice. (C) Acid secretion from mice injected with vehicle (open bar), IL-1β (black bar), omeprazole (grey bar), and omeprazole and IL-1β (open bar) was measured and expressed as μEq of acid H+. Shown is the mean ± SEM. *P < .05 relative to uninfected or vehicle-treated mice. Quantitative reverse-transcription PCR for Shh was performed on total corpus RNA from vehicle (open), IL-1β (filled bar), omeprazole (grey), and omeprazole and IL-1β–treated (open bar) mouse stomachs. (D) Shown is the ratio of Shh mRNA to GAPDH mRNA. The mean ± SEM for 8 mice is shown. *P < .05 compared with vehicle-treated mice. **P < .05 for OM compared with IL-1β plus OM treatment. om, omeprazole.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

7. Figure 6
IL-1R1 is required for inhibition of Shh by IL-1β. Colocalization of (A) H+,K+-ATPase (green) and (B) IL-1β receptor 1 (red). (C) Quantitative reverse-transcription PCR was performed on RNA extracted from organ cultures isolated from gastric corpus of PBS-treated (open bars) or IL-1β–treated (black bars) stomachs isolated from WT or IL-1R1KO mice and treated with PBS (grey bar) or IL-1β (black bar). Shown is the ratio of Shh to GAPDH mRNA expressed as the mean ± SEM for 4 mice. *P < .05 compared with vehicle-treated mice.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

8. Figure 7
IL-1β inhibits parietal cell–specific Shh expression. Primary cultures of canine parietal cells were treated with PBS, interferon (ifn) γ, and IL-1β. (A) Whole-cell lysates were analyzed by immunoblot. Primary cultures of canine mucous cells were treated with PBS and IL-1β. (B) Whole-cell lysates were analyzed by immunoblot. Shown is the mean ± SEM for 3 separate parietal cell and mucous cell preparations. *P < .05 compared with PBS treatment.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

9. Figure 8
IL-1β inhibits acid-dependent release of intracellular calcium. Primary cultures of canine parietal cells preloaded with the Fura 2-AM dye were perfused with (A) Hanks’ balanced salt solution at pH 7 (upper panel), pH 5 (middle panel), or pH 5 with IL-1β (lower panel). (B) Representative measurement of calcium binding to Fura 2 performed 3 times.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

10. Supplementary Figure 1
Helicobacter infection depresses gastric acidity. A Warthin–Starry silver staining of X-gal–stained paraffin sections from mice infected for (A) 3 weeks with H felis and for (C) 8 weeks with H felis. Magnification, 1000×. Changes in gastric acidity at (B) 3 and (D) 8 weeks were expressed as μEq of hydrogen ions (H+).
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

11. Supplementary Figure 2
No change in parietal cell numbers with IL-1β or omeprazole. Confocal images of paraffin sections from mice injected with (A) vehicle, (B) IL-1β, (C) omeprazole, or (D) omeprazole and IL-1β for 3 hours followed by immunohistochemistry for H+-K+-ATPase (red) is shown. 4′,6-diamidino-2-phenylindole was used to stain the nuclei (blue). The insets are a low-power overview at 100×. (E) Morphometric analysis of the total number of parietal cells per gland from mice injected with vehicle, IL-1β, omeprazole, or omeprazole and IL-1β. Shown is the mean ± SEM for 25 glands per mouse. There were 5 mice per group.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions