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Non-vascular myocyte TLR4 regulates intestinal and systemic inflammation and prevents late ileus in murine endotoxemia
Vol. 215, No. 3S, September 2012
signaling is independent from ASC depending inflammasome IL-1b production. CONCLUSIONS: POI is influenced by upstream innate immune activation of MyD88-signaling via IL-1, but not TLR or IL-18. Transient targeting of IL-1-signaling might be an encouraging strategy preventing POI.
Non-vascular myocyte TLR4 regulates intestinal and systemic inflammation and prevents late ileus in murine endotoxemia Bettina M Buchholz, MD, Yoram Vodovotz, PhD, Derek Barclay, Richard A Shapiro, Timothy R Billiar, MD, FACS, Chhinder Sodhi, PhD, David J Hackam, MD, PhD, FACS, Anthony J Bauer, PhD University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Non-hemopoietic TLR4/MyD88-competence drives early murine endotoxin-triggered ileus, while inflammatory non-hemopoietic and hemopoietic synergy results in IL-6Rdependent, TLR4-triggered late ileus. Our aim was to discern the contribution of smooth muscle cell TLR4 activation to inflammation and ileus. METHODS: Selective smooth muscle TLR4-deficient (smooth muscle 22-kDa protein [SM22]-CreⴞTLR4flox/flox) mice and agematched littermates (TLR4flox/flox) were injected i.p. with 5 mg/kg purified lipopolysaccharide (PELPS) followed by a booster PELPSinjection 2 h later. RESULTS: SM22-Cre/loxP smooth muscle specificity was confirmed using fluorescent Cre-reporter mice, and SM22-driven Cre recombinase activity was restricted to nonvascular smooth muscle cells. At 6 h after the first PELPS treatment, SM22-CreⴞTLR4flox/ flox mice displayed severe ileus similar to TLR4flox/flox mice (geometric center [GC] 5.5⫾0.3 vs. 5.1⫾0.6). Still, non-vascular smooth muscle TLR4 signaling differentially regulated enteric and systemic inflammation, with significantly decreased intestinal muscularis IL-6 mRNA (609⫾71.7 vs. 172⫾30.7 fold increase, p⬍0.01) and significantly diminished circulating IL-6 protein (59572⫾10763.8 vs. 10874⫾2102.8 pg/mL, p⬍0.01) but unchanged enteric KC mRNA and circulating protein (141685⫾ 11843.3 vs. 128743⫾11425.2 pg/mL, n.s.). Hence, SM22CreⴞTLR4flox/flox mice recovered from sickness behavior and ileus at 24 h (GC 9.6⫾0.2), with systemic levels of IL-6 protein (139⫾22.6 pg/mL) and KC protein (2165⫾353.7 pg/mL) near baseline. In contrast, TLR4flox/flox mice exhibited sustained ileus (GC 5.0⫾0.9, p⬍0.05) and elevated circulating IL-6 (15879⫾ 6277.6 pg/mL, n.s.) and KC protein (117494⫾25865.2 pg/mL, p⬍0.01). CONCLUSIONS: TLR4-activated non-vascular smooth muscle cells are potent regulators of enteric and systemic inflammation. Reduced smooth muscle-derived, TLR4-triggered inflammation does not protect from early non-hemopoietic ileus, but consequently prevents mediator-driven late ileus.
Abstracts
S23
Endogenous interleukin (IL)-23 protects against acinar injury in lipopolysaccharide (LPS)-mediated mouse severe acute pancreatitis Gregory M Boucher, DO, Albert E Stanek, MD, Sidharth Sharma, MD, Cathy M Mueller, BSc(Hons), Gainosuke Sugiyama, MD, Thomas K Weber, MD, FACS, Nico Ghilardi, PhD, Wenjun Ouyang, PhD, Antonio E Alfonso, MD, FACS, Chongmin Huan, MD, PhD State University of New York, Downstate Medical Center, Brooklyn, NY, VA New York Harbor Health Care System Brooklyn, NY, Genentech, Inc. South San Francisco, CA INTRODUCTION: Severe acute pancreatitis (SAP) remains a significant clinical cause of mortality. Lipopolysaccharide (LPS) appears relevant to the severity of AP in patients and animal models. Animal studies using different AP models have shown that LPS produces diverse effects on the pancreas involving inflammation and acinar damage or protection via unclear mechanisms. Interleukin-23 (IL23) can be derived from LPS stimulated myeloid cells and plays a key role in multiple inflammatory diseases. Depending on the organ and tissue, IL-23 can be harmful or protective in the development of disease. Studies in both patients and animals have reported that serum levels of IL-23 increase in AP with unknown function. To characterize the role of IL-23 in SAP and its relationship with LPS in acinar damage, we studied LPS and cerulein-induced SAP in IL-23/ p19 knockout mice. METHODS: SAP was induced with seven intraperitoneal injections of cerulein (50g/kg) and one intraperitoneal injection of LPS (7mg/kg). 16 hours after treatment, serum and pancreatic tissues were taken for molecular, biochemical and histological studies of pancreatic inflammation and acinar injury. RESULTS: The expression of IL-23 receptor was detected in the mouse pancreas. Compared to WT mice, IL-23/p19 knockout mice demonstrated more destructive SAP as evidenced by larger scale acinar necrosis, more inflammatory infiltration and higher elevated serum amylase levels (p⬍0.05). Consistently, administration of LPS alone was sufficient to cause mild pancreatic injury in IL-23/p19 knockout mice, but not in WT mice. CONCLUSIONS: Our results indicate that IL-23 plays a tissue protective role in LPS-mediated mouse SAP.
Cerulein-treated Ras-activated mice develop painful chronic pancreatitis Sandhya Bondada, MD, Cali Johnson, BA, Fiore Cattaruzza, PhD, Wendy Cedron, BA, Eileen Grady, PhD, Baoan Ji, PhD, Craig Logsdon, PhD, Kimberly S Kirkwood, MD, FACS University of California San Francisco, San Francisco, CA INTRODUCTION: The majority of human pancreatic cancers are associated with activating mutations in the K-ras oncogene that result in constitutive expression of proinflammatory cytokines via NF-kB signaling. It has been recently shown that targeted expression K-rasG12D in acinar cells can lead to ductal adenocarcinoma, but only after a second “hit”, such as the induction of mild chronic pancreatitis. We hypothesized that the augmentation of cerulein-
signaling is independent from ASC depending inflammasome IL-1b production. CONCLUSIONS: POI is influenced by upstream innate immune activation of MyD88-signaling via IL-1, but not TLR or IL-18. Transient targeting of IL-1-signaling might be an encouraging strategy preventing POI.
Non-vascular myocyte TLR4 regulates intestinal and systemic inflammation and prevents late ileus in murine endotoxemia Bettina M Buchholz, MD, Yoram Vodovotz, PhD, Derek Barclay, Richard A Shapiro, Timothy R Billiar, MD, FACS, Chhinder Sodhi, PhD, David J Hackam, MD, PhD, FACS, Anthony J Bauer, PhD University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Non-hemopoietic TLR4/MyD88-competence drives early murine endotoxin-triggered ileus, while inflammatory non-hemopoietic and hemopoietic synergy results in IL-6Rdependent, TLR4-triggered late ileus. Our aim was to discern the contribution of smooth muscle cell TLR4 activation to inflammation and ileus. METHODS: Selective smooth muscle TLR4-deficient (smooth muscle 22-kDa protein [SM22]-CreⴞTLR4flox/flox) mice and agematched littermates (TLR4flox/flox) were injected i.p. with 5 mg/kg purified lipopolysaccharide (PELPS) followed by a booster PELPSinjection 2 h later. RESULTS: SM22-Cre/loxP smooth muscle specificity was confirmed using fluorescent Cre-reporter mice, and SM22-driven Cre recombinase activity was restricted to nonvascular smooth muscle cells. At 6 h after the first PELPS treatment, SM22-CreⴞTLR4flox/ flox mice displayed severe ileus similar to TLR4flox/flox mice (geometric center [GC] 5.5⫾0.3 vs. 5.1⫾0.6). Still, non-vascular smooth muscle TLR4 signaling differentially regulated enteric and systemic inflammation, with significantly decreased intestinal muscularis IL-6 mRNA (609⫾71.7 vs. 172⫾30.7 fold increase, p⬍0.01) and significantly diminished circulating IL-6 protein (59572⫾10763.8 vs. 10874⫾2102.8 pg/mL, p⬍0.01) but unchanged enteric KC mRNA and circulating protein (141685⫾ 11843.3 vs. 128743⫾11425.2 pg/mL, n.s.). Hence, SM22CreⴞTLR4flox/flox mice recovered from sickness behavior and ileus at 24 h (GC 9.6⫾0.2), with systemic levels of IL-6 protein (139⫾22.6 pg/mL) and KC protein (2165⫾353.7 pg/mL) near baseline. In contrast, TLR4flox/flox mice exhibited sustained ileus (GC 5.0⫾0.9, p⬍0.05) and elevated circulating IL-6 (15879⫾ 6277.6 pg/mL, n.s.) and KC protein (117494⫾25865.2 pg/mL, p⬍0.01). CONCLUSIONS: TLR4-activated non-vascular smooth muscle cells are potent regulators of enteric and systemic inflammation. Reduced smooth muscle-derived, TLR4-triggered inflammation does not protect from early non-hemopoietic ileus, but consequently prevents mediator-driven late ileus.
Abstracts
S23
Endogenous interleukin (IL)-23 protects against acinar injury in lipopolysaccharide (LPS)-mediated mouse severe acute pancreatitis Gregory M Boucher, DO, Albert E Stanek, MD, Sidharth Sharma, MD, Cathy M Mueller, BSc(Hons), Gainosuke Sugiyama, MD, Thomas K Weber, MD, FACS, Nico Ghilardi, PhD, Wenjun Ouyang, PhD, Antonio E Alfonso, MD, FACS, Chongmin Huan, MD, PhD State University of New York, Downstate Medical Center, Brooklyn, NY, VA New York Harbor Health Care System Brooklyn, NY, Genentech, Inc. South San Francisco, CA INTRODUCTION: Severe acute pancreatitis (SAP) remains a significant clinical cause of mortality. Lipopolysaccharide (LPS) appears relevant to the severity of AP in patients and animal models. Animal studies using different AP models have shown that LPS produces diverse effects on the pancreas involving inflammation and acinar damage or protection via unclear mechanisms. Interleukin-23 (IL23) can be derived from LPS stimulated myeloid cells and plays a key role in multiple inflammatory diseases. Depending on the organ and tissue, IL-23 can be harmful or protective in the development of disease. Studies in both patients and animals have reported that serum levels of IL-23 increase in AP with unknown function. To characterize the role of IL-23 in SAP and its relationship with LPS in acinar damage, we studied LPS and cerulein-induced SAP in IL-23/ p19 knockout mice. METHODS: SAP was induced with seven intraperitoneal injections of cerulein (50g/kg) and one intraperitoneal injection of LPS (7mg/kg). 16 hours after treatment, serum and pancreatic tissues were taken for molecular, biochemical and histological studies of pancreatic inflammation and acinar injury. RESULTS: The expression of IL-23 receptor was detected in the mouse pancreas. Compared to WT mice, IL-23/p19 knockout mice demonstrated more destructive SAP as evidenced by larger scale acinar necrosis, more inflammatory infiltration and higher elevated serum amylase levels (p⬍0.05). Consistently, administration of LPS alone was sufficient to cause mild pancreatic injury in IL-23/p19 knockout mice, but not in WT mice. CONCLUSIONS: Our results indicate that IL-23 plays a tissue protective role in LPS-mediated mouse SAP.
Cerulein-treated Ras-activated mice develop painful chronic pancreatitis Sandhya Bondada, MD, Cali Johnson, BA, Fiore Cattaruzza, PhD, Wendy Cedron, BA, Eileen Grady, PhD, Baoan Ji, PhD, Craig Logsdon, PhD, Kimberly S Kirkwood, MD, FACS University of California San Francisco, San Francisco, CA INTRODUCTION: The majority of human pancreatic cancers are associated with activating mutations in the K-ras oncogene that result in constitutive expression of proinflammatory cytokines via NF-kB signaling. It has been recently shown that targeted expression K-rasG12D in acinar cells can lead to ductal adenocarcinoma, but only after a second “hit”, such as the induction of mild chronic pancreatitis. We hypothesized that the augmentation of cerulein-