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Macrophage-Derived Indian Hedgehog Drives Epithelial Proliferation During Regeneration in Response to Injury
of the tested antacids showed that ANT 1 induced dewetting almost immediately after its application to the substrate whereas the other three antacids showed stable layers for at least 30 minutes. The results were similar on glass substrates and in ex-vivo porcine oesophageal mucosa. The acid penetration resistance for the chemical (acid neutralisation capacity) and physical (thickness and stability of the layer) resistance of an antacid layer were best for HTL and ANT 1. The stability of the antacid film however was the lowest for ANT 1. Conclusion: 1) We established novel methods for the quantitative assessment of antacids interaction with oesophageal mucosa. 2) From four studied antacids HTL forms the most stable antacid layer both on glass substrate and on ex vivo porcine oesophageal mucosa and has at the same time the best acid penetration resistance which might be of particular benefit in vivo.
Tu1310
We have previously shown an increase in human esophageal myofibroblasts (HEMFs) in GERD biopsies and an increase in HEMF secretion of inflammatory cytokine IL-6 in response to luminal stimuli such as TLR4 ligand LPS. Epithelial thickness has been reported as a histologic marker of GERD. These observations suggested a novel role for HEMFs in GERD in the human esophagus and inform our hypothesis that HEMF derived paracrine mediators affect the squamous epithelium. Our aim therefore was to use primary and immortalized HEMFs previously established and characterized in our lab to determine the effect of LPS stimulated HEMF IL-6 secretion on squamous epithelium proliferation and differentiation. We utilized two models that recapitulate stratified squamous epithelium: the air-liquid interface (ALI) model in which upper chamber epithelial cells are co-cultured with conditioned media in the lower chamber to determine the effect of HEMF secreted factors on the epithelium; and the 3D organotypic (3D OTC) model in which collagen matrix embedded HEMFs are exposed to LPS prior to epithelial cell seeding. Epithelial cells are not exposed to LPS in either model. Methods: ALI (epithelial cells co-cultured at the time of stratification with control media, recombinant IL-6 (rIL-6), conditioned serum-free HEMF media (cSFMM) and LPS treated cSFMM with and without IL-6 neutralizing antibody (IL-6 nAb)) and 3D OTC (epithelial cells seeded atop control or LPS treated collagen/matrigel embedded HEMFs) models were used to study the effect of LPS stimulated HEMF secretion on epithelial thickness, basal p63, and end target pStat3 with IHC and immunoblot. Results: In the ALI model, basal epithelial layer thickness (7.7 µm vs. 13.8 µm, p< 0.05), basal p63, and pStat3 expression increase with rIL-6 vs. control media. LPS cSFMM in the lower chamber results in an increase in eosin stained suprabasal differentiated layer of the epithelium in the upper chamber. An increase in epithelial pStat3 expression is observed by IHC and immunoblot. Addition of IL-6 nAb to the lower chamber results in a decrease in the basal cell layer of the epithelium with a persistently prominent differentiated layer. In 3D OTC, treatment of collagen embedded HEMFs with LPS increases HEMF IL-6 secretion more than two fold, p< 0.001. Total (24.3 µm vs. 52.9 µm, p<0.05) and basal epithelial thickness (8.1 µm vs. 17.6 µm, p<0.05) increase with LPS treated vs. control HEMFs. Immunoblot and IHC demonstrate an increase in epithelial pStat3 expression. Conclusions: 1. In the ALI and 3D OTC models, observed epithelial morphologic changes and the increase in epithelial thickness with LPS treated HEMFs are mediated at least partially via HEMF derived IL-6; 2. These findings suggest that HEMF IL-6 secretion in response to GERD stimuli may regulate epithelial inflammation and proliferation.
Tu1309 BILE REFLUX INCREASED THE INCIDENCE OF HYPERGASTRINEMIA Mumtaz G. Tabbaa, Christopher Decker Introduction: Gastrin is the primary stimulator of the acid secretion, but the clinical use of serum gastrin has been rarely used outside of the diagnosis of gastrinoma syndrome. The measurement of gastric pH is considered helpful monitor of the efficacy of PPIs and H2 blockers. Hypergastrinemia can be induced by the chronic PPI/H2 blockers treatment, it can complicate the course of the treatment, and possibly increases the risk malignancy [1]. The higher PH of the bile liquid prevents relevant measurement of a PPIs effectiveness using the gastric PH in patients with bile reflux. Since the gastrin hormone release is profoundly influenced by the gastric pH, this study was designed to find the incidence of hypergastrinemia related to the chronic use of acid suppression medications especially in patients with bile reflux. Methods: This study involved 156 patients with severe dyspepsia from erosive disease [GERD/PUD] who were taking PPIs or H2 blockers chronically (> 6 months). All patients underwent EGD followed immediately by obtaining serum gastrin level because of the severity of their illness. Gastric PH was obtained in all patients who did not have bile reflux. Patients with therapeutic PH(>4) were not involved. 45 patients were not taking acid suppression medications divided into two groups based on the presence of bile reflux. 111 patients were taking PPIs or H2 blockers chronically divided also into two groups based on the presence of bile reflux. Patients who had atrophic gastritis, current H. pylori gastritis, gastric surgery, CRI, malignancy, and gastrinoma were excluded. The incidence of Hypergastrinemia was compared among the groups. Results: No hypergastrinemia (HG) found in patients without anti-acid treatment and without bile reflux. 2/25(8%) had HG in patients with bile reflux and without PPI/H2 treatment. 9/55 (16%) had HG in patients with PPI/ H2 treatment but without bile reflux. 27/56 (48%) had HG in patients with PPI/H2 treatment and had bile reflux, p<0.05. Figure 1. Discussion and conclusions: Bile reflux into the stomach was associated significantly with a higher incidence of Hypergastrinemia, the incidence increased three-folds. The incidence of hypergastrinemia can not be attributed to the chronic use of PPI/H2 without the knowledge of bile reflux presence. Hypergastrinemia occurred in the presence of bile reflux without PPI/H2 treatment. Hypergastrinemia from bile reflux could result from direct or indirect stimulation, in addition to this stimulation, increased bile flow into the gut is stimulated by a higher level of gastrin hormone. This association between Hypergastrinemia and bile reflux phenomenon is observed in humans in this study, further studies are needed, including animal model to isolate the mechanisms of bile liquid stimulation of gastrin hormone secretion. [1]S.Dacha et, al. Gastroenterol. Rep.(2015) doi: 10.1093 Incidence of Hypergastrinemia
Tu1311 MACROPHAGE-DERIVED INDIAN HEDGEHOG DRIVES EPITHELIAL PROLIFERATION DURING REGENERATION IN RESPONSE TO INJURY Emma L. Teal, Nina Bertaux-Skeirik, Rui Feng, Jenny Chen, Mark Wunderlich, Loryn L. Holokai, Jayati Chakrabarti, Julie Chang, Nambirajan Sundaram, Jennifer Hawkins, Joseph E. Qualls, Michael Helmrath, Tayyab Diwan, James C. Mulloy, Mario Medvedovic, Yana Zavros Background: Gastric repair in response to injury is regulated by Hedgehog (Hh) ligands. Sonic Hedgehog (Shh) is induced during gastric injury and is known to play a key role in regeneration of the epithelium. Indian Hedgehog (Ihh) mediates gastric epithelial cell proliferation. Infiltrating macrophages within the ulcer express Ihh. Hypothesis: Macrophage-derived Ihh promotes epithelial proliferation during gastric regeneration. Methods: Ulcers were induced in the stomachs of Patched (Ptch)/LacZ transgenic reporter mice using acetic acid-induced injury. Stomachs were collected 1, 3, 5 and 7-days post-ulcer induction and b-galactosidase activity analyzed. The "humanized" (huNRGS) mice were generated in which NRGS mice were engrafted with human umbilical cord blood. Ulcers were induced in huNRGS or non-human (non-engrafted) NRGS mice and the immune phenotype analyzed by CyTOF. RNA sequencing was performed on sorted CD163+ macrophages and gene profile compared to human naïve, M1- and M2-polarized cultured cells. The role of macrophage-derived Ihh as a regulator of gastric epithelial proliferation and regeneration was studied in a novel scratch-wound assay using a co-culture of human bone marrow-derived polarized macrophages and human-derived primary gastric epithelium treated with Smoothened inhibitor vismodegib. Human bone marrow-derived macrophages were isolated from huNRGS mice. 3D organoids derived from human stomach were transferred to a 2D monolayer culture. Changes in epithelial proliferation were measured by immunofluorescence. Changes in secreted Ihh was measured by ELISA. Results: 1) Hedgehog receptor Ptch is induced in response to injury: In the stomachs of Ptch/LacZ transgenic reporter mice, Ptch expression was induced within the epithelium at the ulcer margin, 3-days post-injury. Ptch was also expressed within gastric ulcers of patients. 2) CD163+ cells exhibit a unique phenotype: HuNRGS mice exhibited a significant infiltration of CD163+ macrophages within the stomach 3-days post injury. RNA sequencing revealed that the phenotype of sorted CD163+ macrophages from huNRGS mice was unique from polarized M1 and M2 cultured human macrophages. Among the genes enriched in CD163+ huNRGS macrophages were matrix metalloproteinases and pepsinogen. 3) Macrophage-derived Ihh mediates epithelial proliferation: In a 2D primary human gastric epithelial/macrophage co-culture, M2 macrophages induced epithelial proliferation and Ptch expression at the wound site, that correlated with secretion of Ihh. A response that was blocked with vismodegib treatment. Conclusion: Macrophage-derived Ihh promotes regeneration of the gastric epithelium by inducing epithelial proliferation. These studies may serve as a foundation for the use of Hh-mediated interactions for therapeutic advantage.
S-885
AGA Abstracts
AGA Abstracts
HUMAN ESOPHAGEAL MYOFIBROBLAST IL-6 SECRETION REGULATES SQUAMOUS EPITHELIAL THICKNESS Anisa Shaker, Chao Niu
Tu1310
We have previously shown an increase in human esophageal myofibroblasts (HEMFs) in GERD biopsies and an increase in HEMF secretion of inflammatory cytokine IL-6 in response to luminal stimuli such as TLR4 ligand LPS. Epithelial thickness has been reported as a histologic marker of GERD. These observations suggested a novel role for HEMFs in GERD in the human esophagus and inform our hypothesis that HEMF derived paracrine mediators affect the squamous epithelium. Our aim therefore was to use primary and immortalized HEMFs previously established and characterized in our lab to determine the effect of LPS stimulated HEMF IL-6 secretion on squamous epithelium proliferation and differentiation. We utilized two models that recapitulate stratified squamous epithelium: the air-liquid interface (ALI) model in which upper chamber epithelial cells are co-cultured with conditioned media in the lower chamber to determine the effect of HEMF secreted factors on the epithelium; and the 3D organotypic (3D OTC) model in which collagen matrix embedded HEMFs are exposed to LPS prior to epithelial cell seeding. Epithelial cells are not exposed to LPS in either model. Methods: ALI (epithelial cells co-cultured at the time of stratification with control media, recombinant IL-6 (rIL-6), conditioned serum-free HEMF media (cSFMM) and LPS treated cSFMM with and without IL-6 neutralizing antibody (IL-6 nAb)) and 3D OTC (epithelial cells seeded atop control or LPS treated collagen/matrigel embedded HEMFs) models were used to study the effect of LPS stimulated HEMF secretion on epithelial thickness, basal p63, and end target pStat3 with IHC and immunoblot. Results: In the ALI model, basal epithelial layer thickness (7.7 µm vs. 13.8 µm, p< 0.05), basal p63, and pStat3 expression increase with rIL-6 vs. control media. LPS cSFMM in the lower chamber results in an increase in eosin stained suprabasal differentiated layer of the epithelium in the upper chamber. An increase in epithelial pStat3 expression is observed by IHC and immunoblot. Addition of IL-6 nAb to the lower chamber results in a decrease in the basal cell layer of the epithelium with a persistently prominent differentiated layer. In 3D OTC, treatment of collagen embedded HEMFs with LPS increases HEMF IL-6 secretion more than two fold, p< 0.001. Total (24.3 µm vs. 52.9 µm, p<0.05) and basal epithelial thickness (8.1 µm vs. 17.6 µm, p<0.05) increase with LPS treated vs. control HEMFs. Immunoblot and IHC demonstrate an increase in epithelial pStat3 expression. Conclusions: 1. In the ALI and 3D OTC models, observed epithelial morphologic changes and the increase in epithelial thickness with LPS treated HEMFs are mediated at least partially via HEMF derived IL-6; 2. These findings suggest that HEMF IL-6 secretion in response to GERD stimuli may regulate epithelial inflammation and proliferation.
Tu1309 BILE REFLUX INCREASED THE INCIDENCE OF HYPERGASTRINEMIA Mumtaz G. Tabbaa, Christopher Decker Introduction: Gastrin is the primary stimulator of the acid secretion, but the clinical use of serum gastrin has been rarely used outside of the diagnosis of gastrinoma syndrome. The measurement of gastric pH is considered helpful monitor of the efficacy of PPIs and H2 blockers. Hypergastrinemia can be induced by the chronic PPI/H2 blockers treatment, it can complicate the course of the treatment, and possibly increases the risk malignancy [1]. The higher PH of the bile liquid prevents relevant measurement of a PPIs effectiveness using the gastric PH in patients with bile reflux. Since the gastrin hormone release is profoundly influenced by the gastric pH, this study was designed to find the incidence of hypergastrinemia related to the chronic use of acid suppression medications especially in patients with bile reflux. Methods: This study involved 156 patients with severe dyspepsia from erosive disease [GERD/PUD] who were taking PPIs or H2 blockers chronically (> 6 months). All patients underwent EGD followed immediately by obtaining serum gastrin level because of the severity of their illness. Gastric PH was obtained in all patients who did not have bile reflux. Patients with therapeutic PH(>4) were not involved. 45 patients were not taking acid suppression medications divided into two groups based on the presence of bile reflux. 111 patients were taking PPIs or H2 blockers chronically divided also into two groups based on the presence of bile reflux. Patients who had atrophic gastritis, current H. pylori gastritis, gastric surgery, CRI, malignancy, and gastrinoma were excluded. The incidence of Hypergastrinemia was compared among the groups. Results: No hypergastrinemia (HG) found in patients without anti-acid treatment and without bile reflux. 2/25(8%) had HG in patients with bile reflux and without PPI/H2 treatment. 9/55 (16%) had HG in patients with PPI/ H2 treatment but without bile reflux. 27/56 (48%) had HG in patients with PPI/H2 treatment and had bile reflux, p<0.05. Figure 1. Discussion and conclusions: Bile reflux into the stomach was associated significantly with a higher incidence of Hypergastrinemia, the incidence increased three-folds. The incidence of hypergastrinemia can not be attributed to the chronic use of PPI/H2 without the knowledge of bile reflux presence. Hypergastrinemia occurred in the presence of bile reflux without PPI/H2 treatment. Hypergastrinemia from bile reflux could result from direct or indirect stimulation, in addition to this stimulation, increased bile flow into the gut is stimulated by a higher level of gastrin hormone. This association between Hypergastrinemia and bile reflux phenomenon is observed in humans in this study, further studies are needed, including animal model to isolate the mechanisms of bile liquid stimulation of gastrin hormone secretion. [1]S.Dacha et, al. Gastroenterol. Rep.(2015) doi: 10.1093 Incidence of Hypergastrinemia
Tu1311 MACROPHAGE-DERIVED INDIAN HEDGEHOG DRIVES EPITHELIAL PROLIFERATION DURING REGENERATION IN RESPONSE TO INJURY Emma L. Teal, Nina Bertaux-Skeirik, Rui Feng, Jenny Chen, Mark Wunderlich, Loryn L. Holokai, Jayati Chakrabarti, Julie Chang, Nambirajan Sundaram, Jennifer Hawkins, Joseph E. Qualls, Michael Helmrath, Tayyab Diwan, James C. Mulloy, Mario Medvedovic, Yana Zavros Background: Gastric repair in response to injury is regulated by Hedgehog (Hh) ligands. Sonic Hedgehog (Shh) is induced during gastric injury and is known to play a key role in regeneration of the epithelium. Indian Hedgehog (Ihh) mediates gastric epithelial cell proliferation. Infiltrating macrophages within the ulcer express Ihh. Hypothesis: Macrophage-derived Ihh promotes epithelial proliferation during gastric regeneration. Methods: Ulcers were induced in the stomachs of Patched (Ptch)/LacZ transgenic reporter mice using acetic acid-induced injury. Stomachs were collected 1, 3, 5 and 7-days post-ulcer induction and b-galactosidase activity analyzed. The "humanized" (huNRGS) mice were generated in which NRGS mice were engrafted with human umbilical cord blood. Ulcers were induced in huNRGS or non-human (non-engrafted) NRGS mice and the immune phenotype analyzed by CyTOF. RNA sequencing was performed on sorted CD163+ macrophages and gene profile compared to human naïve, M1- and M2-polarized cultured cells. The role of macrophage-derived Ihh as a regulator of gastric epithelial proliferation and regeneration was studied in a novel scratch-wound assay using a co-culture of human bone marrow-derived polarized macrophages and human-derived primary gastric epithelium treated with Smoothened inhibitor vismodegib. Human bone marrow-derived macrophages were isolated from huNRGS mice. 3D organoids derived from human stomach were transferred to a 2D monolayer culture. Changes in epithelial proliferation were measured by immunofluorescence. Changes in secreted Ihh was measured by ELISA. Results: 1) Hedgehog receptor Ptch is induced in response to injury: In the stomachs of Ptch/LacZ transgenic reporter mice, Ptch expression was induced within the epithelium at the ulcer margin, 3-days post-injury. Ptch was also expressed within gastric ulcers of patients. 2) CD163+ cells exhibit a unique phenotype: HuNRGS mice exhibited a significant infiltration of CD163+ macrophages within the stomach 3-days post injury. RNA sequencing revealed that the phenotype of sorted CD163+ macrophages from huNRGS mice was unique from polarized M1 and M2 cultured human macrophages. Among the genes enriched in CD163+ huNRGS macrophages were matrix metalloproteinases and pepsinogen. 3) Macrophage-derived Ihh mediates epithelial proliferation: In a 2D primary human gastric epithelial/macrophage co-culture, M2 macrophages induced epithelial proliferation and Ptch expression at the wound site, that correlated with secretion of Ihh. A response that was blocked with vismodegib treatment. Conclusion: Macrophage-derived Ihh promotes regeneration of the gastric epithelium by inducing epithelial proliferation. These studies may serve as a foundation for the use of Hh-mediated interactions for therapeutic advantage.
S-885
AGA Abstracts
AGA Abstracts
HUMAN ESOPHAGEAL MYOFIBROBLAST IL-6 SECRETION REGULATES SQUAMOUS EPITHELIAL THICKNESS Anisa Shaker, Chao Niu