Publications by authors named "Xinshou Ouyang"

38 Publications

The fermented soy beverage Q-CAN® plus induces beneficial changes in the oral and intestinal microbiome.

BMC Nutr 2021 Mar 4;7(1). Epub 2021 Mar 4.

Department of Pediatrics (General Pediatrics), Yale University School of Medicine, New Haven, USA.

Background: Soy products are associated with many beneficial health consequences, but their effects on the human intestinal microbiome are poorly characterized.

Objectives: To identify the changes in the oral and fecal microbiome in lean and obese participants due to consumption of Q-CAN®, and to assess the expected consequences of these changes based on the published literature.

Methods: Prospective study of lean (10) and obese (9) participants consuming Q-CAN® twice daily for 4 weeks with 8 weeks follow-up. Microbial DNA was extracted from saliva and stool samples, amplified against the V4 region of the 16S ribosomal RNA gene and data analyzed using QIIME 1.9.1 bioinformatics. Four hundred forty-four samples were collected in total, 424 of which were productive and yielded good quality data.

Results: STOOL. In the lean population Bifidobacteria and Blautia show a significant increase while taking Q-CAN®, and there was a trend for this in the obese population. ORAL. There were relatively fewer major changes in the oral microbiome with an increase in the family Veillonellaceae in the lean population while on Q-CAN®.

Conclusion: Q-CAN® consumption induced a number of significant changes in the fecal and oral microbiome. Most notably an increase in the stool microbiome of Bifidobacteria and Blautia, both of which are associated with positive health benefits, and in the saliva an increase in Veillonellaceae.

Trial Registration: This trial was registered with Clinicaltrials.gov on January 14th 2016. ClinicalTrials.gov Identifier: NCT02656056.
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http://dx.doi.org/10.1186/s40795-021-00408-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931600PMC
March 2021

Corrigendum to "EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells".

Int J Inflam 2020 27;2020:3503814. Epub 2020 Sep 27.

Department of Endocrinology, Southwest Hospital of Third Military Medical University, Chongqing, China.

[This corrects the article DOI: 10.1155/2015/301562.].
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http://dx.doi.org/10.1155/2020/3503814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537673PMC
September 2020

Glycogen synthase kinase-3β inhibition alleviates activation of the NLRP3 inflammasome in myocardial infarction.

J Mol Cell Cardiol 2020 12 28;149:82-94. Epub 2020 Sep 28.

Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China. Electronic address:

Inflammasome-promoted sterile inflammation following cardiac damage is critically implicated in heart dysfunction after myocardial infarction (MI). Glycogen synthase kinase-3 (GSK-3β) is a prominent mediator of the inflammatory response, and high GSK-3 activity is associated with various heart diseases. We investigated the regulatory mechanisms of GSK-3β in activation of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in a rat model with successful induction of MI on days 2-28. An in vitro investigation was performed using newborn rat/human cardiomyocytes and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. GSK-3β inhibition markedly improved myocardial dysfunction and prevented remodeling, with parallel reduction in the parameters of NLRP3 inflammasome activation after MI. GSK-3β inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes. GSK-3β's interaction with activating signal cointegrator (ASC) as well as GSK-3β inhibition reduced ASC phosphorylation and oligomerization at the tissues and cellular levels. Taken together, these data show that GSK-3β directly mediates NLRP3 inflammasome activation, causing cardiac dysfunction in MI.
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http://dx.doi.org/10.1016/j.yjmcc.2020.09.009DOI Listing
December 2020

Inflammasome Is Activated in the Liver of Cholestatic Patients and Aggravates Hepatic Injury in Bile Duct-Ligated Mouse.

Cell Mol Gastroenterol Hepatol 2020 27;9(4):679-688. Epub 2019 Dec 27.

Liver Center, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut. Electronic address:

Background & Aims: Inflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study.

Methods: Gene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1 (Casp1) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids.

Results: Caspase-1, NLRP1, NLRP3 and IL-1β were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1β (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1 BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1 BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1 BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1 BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300μM TCA), neither IL-1β induction nor procaspase-1 cleavage were detected.

Conclusions: The inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.
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http://dx.doi.org/10.1016/j.jcmgh.2019.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160576PMC
May 2021

The SGLT2 inhibitor dapagliflozin attenuates the activity of ROS-NLRP3 inflammasome axis in steatohepatitis with diabetes mellitus.

Ann Transl Med 2019 Sep;7(18):429

Department of Endocrinology, Southwest Hospital, the Third Military Medical University (Army Medical University), Chongqing 400038, China.

Background: Diabetes mellitus (DM) is considered as a risk factor for the progress of liver diseases. After tissue damage, there is the highest amplitude of ubiquitously sterile inflammatory response in the liver, resulting in a major clinical consequence concerning a high prevalence of steatohepatitis in DM patients. This study aimed to investigate the inhibitory efficacy of dapagliflozin (DAPA), a sodium glucose cotransporter-2 (SGLT2) inhibitor, on experimental steatohepatitis with DM.

Methods: DM-steatohepatitis model was established by dual intraperitoneal injection of streptozotocin (STZ) and feeding with the high-fat diet (HFD) in apolipoprotein E-deficient (ApoE) mice (n=40). The mice were concurrently treated with DAPA (1 mg/kg/d) by gavage for 12 weeks.

Results: In ApoE mice, dual HFD/STZ dramatically induced hepatic damage and inflammation as compared with HFD alone. DAPA treatment was effective to protect from hepatic damage and inflammation in dual HFD/STZ treated ApoE mice. DAPA also significantly the probability decreased the blood glucose, hepatic lipid accumulation, liver steatosis, and fibrotic response in dual HFD/STZ treated ApoE mice. Further mechanistic investigations indicated that the protection of DAPA on diabetic liver injury was associated with the suppressed production of hepatic reactive oxygen species (ROS) and malondialdehyde (MDA) and the inhibited activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome.

Conclusions: These data demonstrate the efficacy of DAPA for protecting liver damage, inflammation and steatosis from experimental steatohepatitis with DM, and indicate a possible involvement of the inhibited activity of ROS-NLRP3 inflammasome.
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http://dx.doi.org/10.21037/atm.2019.09.03DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803170PMC
September 2019

Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation.

Am J Physiol Gastrointest Liver Physiol 2019 10 14;317(4):G387-G397. Epub 2019 Aug 14.

Section of Digestive Diseases, Yale University School of medicine, New Haven, Connecticut.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH. This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.
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http://dx.doi.org/10.1152/ajpgi.00054.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842989PMC
October 2019

The Reduced Expression of EOLA1 May Be Related to Refractory Diabetic Foot Ulcer.

Mediators Inflamm 2019 17;2019:6705424. Epub 2019 Mar 17.

Department of Endocrinology, First Affiliated Hospital of Army Medical University (Third Military Medical University), Chongqing 400038, China.

Background: Chronic diabetic foot ulcer (DFU) is one of the most intractable complications of diabetes mellitus (DM). Its pathogenesis is complex, and uncontrolled chronic inflammation is an important factor. Endothelial overexpressed lipopolysaccharide-associated factor 1 (EOLA1) discovered in our laboratory is an intracellular protein with the function of inflammatory regulation. This study was aimed at observing the expression of EOLA1 in DFU skin tissues and its relationship with inflammation and at exploring the possible role of EOLA1 in DFU and its mechanism.

Methods: The patients with DFU were divided into 2 groups based on the formation time of ulcer: the acute wound (AW) group with the course of disease ≤ 4 weeks and the chronic wound (CW) group with the course of disease > 4 weeks. The relevant clinical data of patients were collected, and the skin tissues around the ulcer were used for immunofluorescence detection and immunohistochemical staining to observe inflammation. The expression levels of EOLA1, metallothionein 2A (MT2A), nuclear factor-B (NF-B), and interleukin-6 (IL-6) were detected by western blot.

Results: A total of 79 patients were enrolled in the study. The results of immunofluorescence and immunohistochemistry showed that EOLA1 was expressed in the epithelial tissues of DFU. However, the expression of EOLA1 in the CW group was significantly lower than that in the AW group ( < 0.05), and the expression of NF-B and IL-6 was obviously increased ( < 0.05).

Conclusion: The refractory wounds in patients with DFU may be closely related to the uncontrolled activation of inflammatory pathways in cells caused by the reduced expression of negative regulators of inflammation (e.g., EOLA1), and such decreased expression may be also strongly linked to the persistent state of inflammation.
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http://dx.doi.org/10.1155/2019/6705424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441532PMC
August 2019

The multi-dimensional role of intestinal HIFs in liver pathobiology.

J Hepatol 2018 Oct 10;69(4):772-773. Epub 2018 Aug 10.

Section of Digestive Diseases, Yale University, New Haven, CT, United States. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2018.07.012DOI Listing
October 2018

β-Hydroxybutyrate protects from alcohol-induced liver injury via a Hcar2-cAMP dependent pathway.

J Hepatol 2018 09 27;69(3):687-696. Epub 2018 Apr 27.

Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT 06520, USA; USA West Haven Veterans Medical Center, West Haven, CT 06516, USA. Electronic address:

Background & Aims: Sterile inflammation resulting in alcoholic hepatitis (AH) occurs unpredictably after many years of excess alcohol intake. The factors responsible for the development of AH are not known but mitochondrial damage with loss of mitochondrial function are common features. Hcar2 is a G-protein coupled receptor which is activated by β-hydroxybutyrate (BHB). We aimed to determine the relevance of the BHB-Hcar2 pathway in alcoholic liver disease.

Methods: We tested if loss of BHB production can result in increased liver inflammation. We further tested if BHB supplementation is protective in AH through interaction with Hcar2, and analyzed the immune and cellular basis for protection.

Results: Humans with AH have reduced hepatic BHB, and inhibition of BHB production in mice aggravated ethanol-induced AH, with higher plasma alanine aminotransferase levels, increased steatosis and greater neutrophil influx. Conversely supplementation of BHB had the opposite effects with reduced alanine aminotransferase levels, reduced steatosis and neutrophil influx. This therapeutic effect of BHB is dependent on the receptor Hcar2. BHB treatment increased liver Il10 transcripts, and promoted the M2 phenotype of intrahepatic macrophages. BHB also increased the transcriptional level of M2 related genes in vitro bone marrow derived macrophages. This skewing towards M2 related genes is dependent on lower mitochondrial membrane potential (Δψ) induced by BHB.

Conclusions: Collectively, our data shows that BHB production during excess alcohol consumption has an anti-inflammatory and hepatoprotective role through an Hcar2 dependent pathway. This introduces the concept of metabolite-based therapy for AH.

Lay Summary: Alcoholic hepatitis is a life-threatening condition with no approved therapy that occurs unexpectedly in people who consume excess alcohol. The liver makes many metabolites, and we demonstrate that loss of one such metabolite β-hydroxybutyrate occurs in patients with alcoholic hepatitis. This loss can increase alcohol-induced liver injury, and β-hydroxybutyrate can protect from alcohol-induced liver injury via a receptor on liver macrophages. This opens the possibility of metabolite-based therapy for alcoholic hepatitis.
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http://dx.doi.org/10.1016/j.jhep.2018.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098974PMC
September 2018

Digoxin Suppresses Pyruvate Kinase M2-Promoted HIF-1α Transactivation in Steatohepatitis.

Cell Metab 2018 02;27(2):339-350.e3

Section of Digestive Diseases, Yale University, New Haven, CT 06520, USA; West Haven Veterans Medical Center, West Haven, CT 06516, USA. Electronic address:

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.
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http://dx.doi.org/10.1016/j.cmet.2018.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806149PMC
February 2018

Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response.

JCI Insight 2017 03 9;2(5):e90780. Epub 2017 Mar 9.

Department of Internal Medicine and Yale Liver Center, Yale University School of Medicine, New Haven, Connecticut, USA.

Mechanisms of bile acid-induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies. We examined how BAs initiate liver injury using isolated liver cells from humans and mice and in-vivo mouse models. At pathophysiologic concentrations, BAs induced proinflammatory cytokine expression in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes. These hepatocyte-specific cytokines stimulated neutrophil chemotaxis. Inflammatory injury was mitigated in mice treated with BA or after bile duct ligation, where less hepatic infiltration of neutrophils was detected. Neutrophils in periportal areas of livers from cholestatic patients also correlated with elevations in their serum aminotransferases. This liver-specific inflammatory response required BA entry into hepatocytes via basolateral transporter Ntcp. Pathophysiologic levels of BAs induced markers of ER stress and mitochondrial damage in mouse hepatocytes. Chemokine induction by BAs was reduced in hepatocytes from mice, while liver injury was diminished both in conventional and hepatocyte-specific mice, confirming a role for Tlr9 in BA-induced liver injury. These findings reveal potentially novel mechanisms whereby BAs elicit a hepatocyte-specific cytokine-induced inflammatory liver injury that involves innate immunity and point to likely novel pathways for treating cholestatic liver disease.
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http://dx.doi.org/10.1172/jci.insight.90780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333973PMC
March 2017

The SGLT-2 Inhibitor Dapagliflozin Has a Therapeutic Effect on Atherosclerosis in Diabetic ApoE Mice.

Mediators Inflamm 2016 26;2016:6305735. Epub 2016 Dec 26.

Department of Endocrinology, The First Affiliated Hospital of Third Military Medical University, Chongqing 400038, China.

. Our study aimed to observe the effect of sodium glucose cotransporter-2 (SGLT2) inhibitor dapagliflozin on diabetic atherosclerosis and investigate the subsequent mechanism. . Aortic atherosclerosis was induced in streptozotocin induced diabetic ApoE mice by feeding with high-fat diet, and dapagliflozin was administrated intragastrically for 12 weeks as treatment. Effects of dapagliflozin on indices of glucose and fat metabolism, IL-1, IL-18, NLRP3 protein levels, and the reactive oxygen species (ROS) were measured. The atherosclerosis was evaluated by oil red O and hematoxylin-eosin staining. The effects of dapagliflozin on the IL-1 production in culturing primary macrophages of wild type and NLRP3 knockout mice were investigated for mechanism analyses. . Dapagliflozin treatment showed favorable effects on glucose and fat metabolism, partially reversed the formation of atherosclerosis, inhibited macrophage infiltration, and enhanced the stability of lesion. Also, reduced production of IL-1, IL-18, NLRP3 protein, and mitochondrial ROS in the aortic tissues was detected with dapagliflozin treatment. In vitro, NLRP3 inflammasome was activated by hyperglucose and hyperlipid through ROS pathway. . Dapagliflozin may be of therapeutic potential for diabetic atherosclerosis induced by high-fat diet, and these benefits may depend on the inhibitory effect on the secretion of IL-1 by macrophages via the ROS-NLRP3-caspase-1 pathway.
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http://dx.doi.org/10.1155/2016/6305735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220517PMC
August 2017

The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury.

Science 2016 11;354(6313):765-768

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure.
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http://dx.doi.org/10.1126/science.aaf7532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640175PMC
November 2016

EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells.

Int J Inflam 2015 31;2015:301562. Epub 2015 Dec 31.

Department of Endocrinology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.

Our research group firstly discovered endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1, GenBank number AY074889) as a lipopolysaccharide (LPS) responsive gene in ECV304 cells. The previous studies have further demonstrated the association of EOLA1 with metallothionein 2A (MT2A), while the role of EOLA1 during LPS-induced inflammatory response in ECV304 cells is unknown. In this report, we determined the subcellular localization of EOLA1 and the regulatory capacity of EOLA1 on vascular cell adhesion molecule-1 (VCAM-1) in response to LPS in ECV304 cells. Our results show that EOLA1 is broadly diffuse in the cells, and EOLA1 expression is dramatically induced by LPS. EOLA1 knockdown results in significant enhancement of LPS-induced VCAM-1 production. Consistent with this, overexpression of EOLA1 leads to the reduction of LPS-induced VCAM-1 production. Furthermore, MT2A knockdown reduces LPS-induced VCAM-1 production. Collectively, our results demonstrate a negative regulatory role of EOLA1 on LPS-induced VCAM-1 expression involving its association with MT2A in ECV304 cells.
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http://dx.doi.org/10.1155/2015/301562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736203PMC
February 2016

Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.

J Clin Invest 2016 Mar 25;126(3):859-64. Epub 2016 Jan 25.

Nonalcoholic steatohepatitis (NASH) is the most common liver disease in industrialized countries. NASH is a progressive disease that can lead to cirrhosis, cancer, and death, and there are currently no approved therapies. The development of NASH in animal models requires intact TLR9, but how the TLR9 pathway is activated in NASH is not clear. Our objectives in this study were to identify NASH-associated ligands for TLR9, establish the cellular requirement for TLR9, and evaluate the role of obesity-induced changes in TLR9 pathway activation. We demonstrated that plasma from mice and patients with NASH contains high levels of mitochondrial DNA (mtDNA) and intact mitochondria and has the ability to activate TLR9. Most of the plasma mtDNA was contained in microparticles (MPs) of hepatocyte origin, and removal of these MPs from plasma resulted in a substantial decrease in TLR9 activation capacity. In mice, NASH development in response to a high-fat diet required TLR9 on lysozyme-expressing cells, and a clinically applicable TLR9 antagonist blocked the development of NASH when given prophylactically and therapeutically. These data demonstrate that activation of the TLR9 pathway provides a link between the key metabolic and inflammatory phenotypes in NASH.
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http://dx.doi.org/10.1172/JCI83885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767345PMC
March 2016

Identification of a novel de novo GATA3 mutation in a patient with HDR syndrome.

J Int Med Res 2015 Oct 12;43(5):718-24. Epub 2015 Aug 12.

Department of Endocrinology, First Affiliated Hospital of Third Military Medical University, Chongqing, China

We describe the case of a 21-year-old male with hypocalcaemia, hyperphosphataemia, recurrent limb twitch, deafness, proteinuria, increased serum creatinine and urea nitrogen levels, and shrinkage of both kidneys. Brain computed tomography showed intracranial calcifications. The patient was diagnosed with hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. DNA sequence analysis of the GATA3 gene showed a novel de novo mutation, c. 529dupC (p. Arg177profs*126), in exon 2, resulting in a frameshift mutation with a premature stop codon after a new 126 amino acid sequence. We provide further evidence that HDR syndrome is caused by haploinsufficiency of GATA3.
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http://dx.doi.org/10.1177/0300060515591065DOI Listing
October 2015

Na(+) /H(+) exchanger regulatory factor 1 knockout mice have an attenuated hepatic inflammatory response and are protected from cholestatic liver injury.

Hepatology 2015 Oct 22;62(4):1227-36. Epub 2015 Aug 22.

Yale Liver Center, Yale University School of Medicine, New Haven, CT.

Unlabelled: The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice.

Conclusion: NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases.
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http://dx.doi.org/10.1002/hep.27956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589453PMC
October 2015

Activation of N-methyl-d-aspartate receptor downregulates inflammasome activity and liver inflammation via a β-arrestin-2 pathway.

Am J Physiol Gastrointest Liver Physiol 2014 Oct 7;307(7):G732-40. Epub 2014 Aug 7.

Section of Digestive Diseases, Yale University, New Haven, Connecticut; Section of Digestive Diseases, Department of Veterans Affairs Connecticut Healthcare, West Haven, Connecticut; and

Activation of the cytosolic inflammasome machinery is responsible for acute and chronic liver inflammation, but little is known about its regulation. The N-methyl-d-aspartate (NMDA) receptor families are heterotetrameric ligand-gated ion channels that are activated by a range of metabolites, including aspartate, glutamate, and polyunsaturated fatty acids. In the brain NMDA receptors are present on neuronal and nonneuronal cells and regulate a diverse range of functions. We tested the role of the NMDA receptor and aspartate in inflammasome regulation in vitro and in models of acute hepatitis and pancreatitis. We demonstrate that the NMDA receptor is present on Kupffer cells, and their activation on primary mouse and human cells limits inflammasome activation by downregulating NOD-like receptor family, pyrin domain containing 3 and procaspase-1. The NMDA receptor pathway is active in vivo, limits injury in acute hepatitis, and can be therapeutically further activated by aspartate providing protection in acute inflammatory liver injury. Downregulation of inflammasome activation by NMDA occurs via a β-arrestin-2 NF-kβ and JNK pathway and not via Ca(2+) mobilization. We have identified the NMDA receptor as a regulator of inflammasome activity in vitro and in vivo. This has identified a new area of immune regulation associated by metabolites that may be relevant in a diverse range of conditions, including nonalcoholic steatohepatitis and total parenteral nutrition-induced immune suppression.
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http://dx.doi.org/10.1152/ajpgi.00073.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187065PMC
October 2014

Effect of osteopontin in regulating bone marrow mesenchymal stem cell treatment of skin wounds in diabetic mice.

Diabetes Metab Res Rev 2014 Sep;30(6):457-66

Department of Endocrinology, Southwest Hospital of Third Military Medical University, Chongqing, China.

Background: We aimed to investigate the role of osteopontin in regulating mesenchymal stem cells transplanted to promote wound healing in diabetic mice.

Methods: The mesenchymal stem cells of osteopontin knock-out (KO) and wild-type (WT) mice were isolated separately for in vitro culture and characterization. A skin wound on the back of mice was established by skin punching. In 27 osteopontin KO male mice, induced diabetes mellitus was via intraperitoneal injection of streptozotocin. 9 normal mice were used as controls. The mice were divided into four groups and injected with Dulbecco's modified Eagle's medium (DMEM) or mesenchymal stem cells via the tail vein: A (diabetic mice injected with DMEM), B (diabetic mice injected with osteopontin KO mesenchymal stem cells), C (diabetic mice injected with WT mesenchymal stem cells), D (normal mice injected with DMEM). The healing times and closure rates of skin wounds were recorded. The microvessel density of healing wounds was measured, and the localized expression of osteopontin was identified by western blotting and immunohistochemistry. The migration of mesenchymal stem cells was observed on normal mice with skin wound injected with mesenchymal stem cells of C57BL6~GFP transgenic mice, which show green fluorescent under UV light.

Results: Compared with normal mice, the healing time of wounds in the mice with diabetes and osteopontin KO was significantly prolonged (p < 0.01). After transplanting osteopontin KO mesenchymal stem cells, the healing time was slightly shorter. Meanwhile, the healing time was significantly shorter after transplanted with WT mesenchymal stem cells and more significant neovascularization at healing wounds (p < 0.05). The expression of osteopontin in local healing wounds after transplantation of WT mesenchymal stem cells was demonstrated with western blotting and immunohistochemistry. After 4 days, the green fluoresces were noted on the wounds of mice injected with mesenchymal stem cells of fluorescent mice.

Conclusions: Mesenchymal stem cells can migrate to wound sites, and osteopontin plays a regulatory role in mesenchymal stem cells promoting the healing of diabetic skin wounds.
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http://dx.doi.org/10.1002/dmrr.2566DOI Listing
September 2014

Adenosine is required for sustained inflammasome activation via the A₂A receptor and the HIF-1α pathway.

Nat Commun 2013 ;4:2909

1] Section of Digestive Diseases, Yale University, TAC S223A, New Haven, Connecticut 06520, USA [2] Section of Digestive Diseases, Department of Veterans Affairs Connecticut Healthcare, West Haven, Connecticut 06516, USA.

Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1β, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A(2A) receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of the lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A(2A) receptor-mediated signalling.
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http://dx.doi.org/10.1038/ncomms3909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895487PMC
July 2014

Immune chaperone gp96 drives the contributions of macrophages to inflammatory colon tumorigenesis.

Cancer Res 2014 Jan 9;74(2):446-59. Epub 2013 Dec 9.

Authors' Affiliations: Departments of Microbiology and Immunology and Pathology and Laboratory Medicine; Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina; and Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut.

Macrophages are important drivers in the development of inflammation-associated colon cancers, but the mechanistic underpinnings for their contributions are not fully understood. Furthermore, Toll-like receptors have been implicated in colon cancer, but their relevant cellular sites of action are obscure. In this study, we show that the endoplasmic reticulum chaperone gp96 is essential in tumor-associated macrophages (TAM) to license their contributions to inflammatory colon tumorigenesis. Mice where gp96 was genetically deleted in a macrophage-specific manner exhibited reduced colitis and inflammation-associated colon tumorigenesis. Attenuation of colon cancer in these mice correlated strikingly with reduced mutation rates of β-catenin, increased efficiency of the DNA repair machinery, and reduced expression of proinflammatory cytokines, including interleukin (IL)-17 and IL-23 in the tumor microenvironment. The genotoxic nature of TAM-associated inflammation was evident by increased expression of genes in the DNA repair pathway. Our work deepens understanding of how TAM promote oncogenesis by altering the molecular oncogenic program within epithelial cells, and it identifies gp96 as a lynchpin chaperone needed in TAM to license their function and impact on expression of critical inflammatory cytokines in colon tumorigenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002507PMC
January 2014

Effect of laparoscopic Roux-en-Y gastric bypass surgery on type 2 diabetes mellitus with hypertension: a randomized controlled trial.

Diabetes Res Clin Pract 2013 Jul 22;101(1):50-6. Epub 2013 May 22.

Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.

Aims: The aim of this study was to evaluate the effect of laparoscopic Roux-en-Y gastric bypass (RYGB) surgery compared with usual care with and without Exenatide therapy in obese people with type 2 diabetes mellitus (T2DM) and hypertension.

Methods: 108 obese T2DM with hypertension were enrolled and randomly allocated to usual care (group A), usual care plus Exenatide (group B), and RYGB surgery (group C). Demographic characteristics, metabolic parameters and cardiac structure/function along with inflammatory cytokines were measured and compared before and after 12 months.

Results: At 12 months, diabetes remission had occurred in no patients in groups A and B versus 90% in group C, and there was a significant decrease in requirement of antihypertensive drugs in group C compared with groups A and B (P<0.05). Other parameters (body mass index, hemoglobin A1c, homeostasis model assessment of insulin resistance, lipids), inflammation index (high sensitivity C-reactive protein, tumor necrosis factor-α, high molecular weight adiponectin) and cardiac structure (left ventricular mass index) were significantly improved in groups B and C, but patients in group C had the greatest degree of improvement (P<0.05).

Conclusion: RYGB surgery improves a number of parameters including cardiovascular function in obese hypertensive people with T2DM. This is likely to be due to, at least in part, an improvement in the abnormal metabolic panel and to reduced inflammation.
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http://dx.doi.org/10.1016/j.diabres.2013.04.005DOI Listing
July 2013

Inflammasome biology in fibrogenesis.

Biochim Biophys Acta 2013 Jul 3;1832(7):979-88. Epub 2013 Apr 3.

Section of Digestive Diseases, Yale University, New Haven, CT, USA; West Haven Veterans Medical Center, New Haven, CT, USA.

Pathogens and sterile insults both result in an inflammatory response. A significant part of this response is mediated by cytosolic machinery termed as the inflammasome which results in the activation and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Both of these are known to result in the activation of an acute inflammatory response, resulting in the production of downstream inflammatory cytokines such as tumor necrosis factor (TNF-α), interferon-gamma (IFN-γ), chemotaxis of immune cells, and induction of tissue injury. Surprisingly this very acute inflammatory pathway is also vital for the development of a full fibrogenic response in a number of organs including the lung, liver, and skin. There is evidence for the inflammasome having a direct role on tissue specific matrix producing cells such as the liver stellate cell, and also indirectly through the activation of resident tissue macrophage populations. The inflammasome requires stimulation of two pathways for full activation, and initiating stimuli include Toll-like receptor (TLR) agonists, adenosine triphosphate (ATP), particulates, and oxidative stress. Such a role for an acute inflammatory pathway in fibrosis runs counter to the prevailing association of TGF-β driven anti-inflammatory and pro-fibrotic pathways. This identifies new therapeutic targets which have the potential to simultaneously decrease inflammation, tissue injury and fibrosis. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
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http://dx.doi.org/10.1016/j.bbadis.2013.03.020DOI Listing
July 2013

Inflammasome components Asc and caspase-1 mediate biomaterial-induced inflammation and foreign body response.

Proc Natl Acad Sci U S A 2011 Dec 22;108(50):20095-100. Epub 2011 Nov 22.

Section of Digestive Diseases and Departments of Pathology and Biomedical Engineering, Yale University, New Haven, CT 06520, USA.

Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.
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http://dx.doi.org/10.1073/pnas.1105152108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3250158PMC
December 2011

Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation.

Nat Commun 2011 ;2:314

Immunology Institute, Department of Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029, USA.

T(H)17 cells are recognized as a unique subset of T helper cells that have critical roles in the pathogenesis of autoimmunity and tissue inflammation. Although RORγt is necessary for the generation of T(H)17 cells, the molecular mechanisms underlying the functional diversity of T(H)17 cells are not fully understood. Here we show that a member of interferon regulatory factor (IRF) family of transcription factors, IRF8, has a critical role in silencing T(H)17-cell differentiation. Mice with a conventional knockout, as well as a T cell-specific deletion, of the Irf8 gene exhibited more efficient T(H)17 cells. Indeed, studies of an experimental model of colitis showed that IRF8 deficiency resulted in more severe inflammation with an enhanced T(H)17 phenotype. IRF8 was induced steadily and inhibited T(H)17-cell differentiation during T(H)17 lineage commitment at least in part through its physical interaction with RORγt. These findings define IRF8 as a novel intrinsic transcriptional inhibitor of T(H)17-cell differentiation.
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http://dx.doi.org/10.1038/ncomms1311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112536PMC
August 2011

Potentiation of Th17 cytokines in aging process contributes to the development of colitis.

Cell Immunol 2011 27;266(2):208-17. Epub 2010 Oct 27.

Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, United States.

Th17 cells, which produce IL-17 and IL-22, promote autoimmunity in mice and have been implicated in the pathogenesis of autoimmune/inflammatory diseases in humans. However, the Th17 immune response in the aging process is still not clear. In the present study, we found that the induction of IL-17-producing CD4(+) T cells was significantly increased in aged individuals compared with young healthy ones. The mRNA expression of IL-17, IL-17F, IL-22, and RORC2 was also significantly increased in aged people. Similar to humans, Th17 cells as well as mRNAs encoding IL-17, IL-22 and RORγt were dramatically elevated in naïve T cells from aged mouse compared to young ones. In addition, CD44 positive IL-17-producing CD4(+) T cells were significantly higher in aged mice, suggesting that memory T cells are an important source of IL-17 production. Furthermore, the percentage of IL-17-producing CD4(+) T cells generated in co-culture with dendritic cells from either aged or young mice did not show significant differences, suggesting that dendritic cells do not play a primary role in the elevation of Th17 cytokines in aged mouse cells. Importantly, transfer of CD4(+)CD45Rb(hi) cells from aged mice induced more severe colitis in RAG(-/-) mice compared to cells from young mice, Taken together, these results suggest that Th17 immune responses are elevated in aging humans and mice and may contribute to the increased development of inflammatory disorders in the elderly.
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http://dx.doi.org/10.1016/j.cellimm.2010.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006034PMC
January 2011

Reduction of Stat3 activity attenuates HIV-induced kidney injury.

J Am Soc Nephrol 2009 Oct 16;20(10):2138-46. Epub 2009 Jul 16.

Department of Nephrology, RuiJin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

HIV-1 Nef induces podocyte proliferation and dedifferentiation by activating the Stat3 and MAPK1,2 pathways. Activation of Stat3 also occurs in human kidneys affected by HIV-associated nephropathy (HIVAN), but its contribution to the development of HIVAN is unknown. Here, we generated HIV-1 transgenic mice (Tg26) with either 75% Stat3 activity (Tg26-SA/+) or 25% Stat3 activity (Tg26-SA/-). The kidneys of Tg26-SA/+ mice, but not Tg26-SA/- mice, showed increased Stat3 phosphorylation. The Tg26-SA/+ phenotype was not different from Tg26 mice, but Tg26-SA/- mice developed significantly less proteinuria, glomerulosclerosis, and tubulointerstitial injury. Tg26-SA/+ mice exhibited reduced expression of podocyte differentiation markers and increased expression of VEGF and proliferation markers as compared to Tg26-SA/- mice. Primary podocytes isolated from Tg26-SA/+ mice showed increased Stat3 phosphorylation and reduced expression of podocyte differentiation markers. The tubulointerstitial compartment and isolated tubules of Tg26-SA/+ mice also had increased Stat3 phosphorylation and expression of Stat3 target genes. We confirmed that the expression of the HIV-1 transgene and reduction of Stat3 activity did not affect T and B cell development. In conclusion, Stat3 plays a critical role in the pathogenesis of HIVAN.
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http://dx.doi.org/10.1681/ASN.2008080879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754106PMC
October 2009

AP-1 activated by toll-like receptors regulates expression of IL-23 p19.

J Biol Chem 2009 Sep 10;284(36):24006-16. Epub 2009 Jul 10.

Immunology Institute, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.

Interleukin (IL)-23, a new member of the IL-12 family, plays a central role in the Th17 immune response and in autoimmune diseases. It is clear that activated macrophages and dendritic cells produce IL-23, but the molecular mechanisms whereby inflammatory signals stimulate IL-23 expression are not fully understood. We demonstrate that induction of IL-23 p19 gene expression by LPS depends on the TLR4 and MyD88 pathways. All three MAPK pathways (ERK, JNK, and p38) that are activated by lipopolysaccharide (LPS) stimulation were shown to exert a positive effect on p19 expression. We cloned a 1.3-kb putative p19 promoter and defined its transcription initiation sites by the 5'-rapid amplification of cDNA ends method. By analyzing IL-23 p19 promoter mutants, we have identified a promoter region (-413 to +10) that contains several important elements, including NF-kappaB and AP-1. In addition to NF-kappaB, we have demonstrated that the proximal AP-1 site is important for p19 promoter activation. Mutation of the AP-1 site resulted in the loss of p19 promoter activation. Electrophoretic mobility shift assay (EMSA) analysis showed that c-Jun and c-Fos bind to the AP-1 site, which was confirmed by a chromatin immunoprecipitation assay. Furthermore, co-transfection of c-Jun and ATF2 synergistically induced p19 promoter activation, and c-Jun and ATF2 formed a protein complex, demonstrated by co-immunoprecipitation. Finally, LPS-stimulated peritoneal macrophages from IL-10-deficient mice expressed significantly higher IL-23 p19 than macrophages from wild type mice, and the addition of recombinant IL-10 strongly inhibited LPS-induced p19 expression. Thus, this study suggests that MyD88-dependent Toll-like receptor signaling induces IL-23 p19 gene expression through both MAPKs and NF-kappaB.
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http://dx.doi.org/10.1074/jbc.M109.025528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781995PMC
September 2009

IFN regulatory factor 8 sensitizes soft tissue sarcoma cells to death receptor-initiated apoptosis via repression of FLICE-like protein expression.

Cancer Res 2009 Feb 20;69(3):1080-8. Epub 2009 Jan 20.

Department of Biochemistry, Medical College of Georgia, Augusta, GA 30912, USA.

IFN regulatory factor 8 (IRF8) has been shown to suppress tumor development at least partly through regulating apoptosis of tumor cells; however, the molecular mechanisms underlying IRF8 regulation of apoptosis are still not fully understood. Here, we showed that disrupting IRF8 function resulted in inhibition of cytochrome c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in soft tissue sarcoma (STS) cells. Inhibition of the mitochondrion-dependent apoptosis signaling cascade is apparently due to blockage of caspase-8 and Bid activation. Analysis of signaling events upstream of caspase-8 revealed that disrupting IRF8 function dramatically increases FLIP mRNA stability, resulting in increased IRF8 protein level. Furthermore, primary myeloid cells isolated from IRF8-null mice also exhibited increased FLIP protein level, suggesting that IRF8 might be a general repressor of FLIP. Nuclear IRF8 protein was absent in 92% (55 of 60) of human STS specimens, and 99% (59 of 60) of human STS specimens exhibited FLIP expression, suggesting that the nuclear IRF8 protein level is inversely correlated with FLIP level in vivo. Silencing FLIP expression significantly increased human sarcoma cells to both FasL-induced and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and ectopic expression of IRF8 also significantly increased the sensitivity of these human sarcoma cells to FasL- and TRAIL-induced apoptosis. Taken together, our data suggest that IRF8 mediates FLIP expression level to regulate apoptosis and targeting IRF8 expression is a potentially effective therapeutic strategy to sensitize apoptosis-resistant human STS to apoptosis, thereby possibly overcoming chemoresistance of STS, currently a major obstacle in human STS therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-2520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633427PMC
February 2009
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