Functionally defective germline variants of sialic acid acetylesterase in autoimmunity.

Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

Gastroenterology, Academic Medicine and a Changing Landscape.

The forces that are reshaping the delivery of health care through much of the developed world are especially acute within academic centers that carry the responsibility for delivering that care while advancing medical knowledge and ensuring well-trained physicians. Gastroenterology will not be spared any of those forces, and in some ways represents the leading edge of their impact. Though the dynamics vary within the context of the health-care delivery and scientific enterprise of individual countries, common elements are demands for greater accountability and transparency in how academic medical centers demonstrate their value while assuring broad access to their expertise. In the United States, underlying many forms of change in the payment scheme are the common elements that will increasingly place the risk for the cost of care on providers rather than on the payers, be it government or private, as has historically been true. At the same time, academic medical centers, with gastroenterology responsible for addressing the burden of digestive diseases, must remain the stem cells for health care integrating all their missions and providing the foundation of medical advances which will ultimately improve human welfare. What will academic gastroenterology units look like if they are able to effectively respond to these forces? Gastrointestinal (GI) divisions and faculty will own new roles including responsibility for system success in caring for patients. They will evolve their training programs to provide the next generation with skills needed to succeed, including the discipline of system improvement, team leadership and others. And there will be new models that will drive the organization of research that are not as conventionally self-contained within the gastroenterology units, but fostering research teams that have hubs and spokes. The vitality of GI divisions will depend on the willingness to seize ownership of the new value proposition of disease management ensuring that each patient achieves the best outcome with the most effective use of resources and endeavor within their systems to capture some of that value to invest in their training and research missions. In the course of that evolution, gastroenterology will be well served by rebalancing the dependence on existing modalities. If procedural gastroenterology becomes the sole value proposition, it will lead to an increasingly narrow view of the field.

Loss of TLR2 worsens spontaneous colitis in MDR1A deficiency through commensally induced pyroptosis.

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b(+) myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1ß activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b(+)Ly6C(+)-derived IL-1ß production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b(+) myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1ß, consistent with pyroptosis. Inhibition of reactive oxygen species-mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis.

Gastroenterology's editors-in-chief: historical and personal perspectives of their editorships.

Fourteen editors-in-chiefs have steered Gastroenterologyto success since its inception in 1943. Five (Alvarez, Ivy, Aaron, Grossman, and Donaldson) are no longer with us. Their personalities and editorships, along with those of Marvin Sleisenger, are presented by their admirers. Fordtran, Ockner, Goyal, LaRusso, Podolsky, Brenner, Rustgi, and Omary describe their own backgrounds, experiences, and personal reflections on serving as editor-in-chief of Gastroenterology.

Increased T-helper 2 cytokines in bile from patients with IgG4-related cholangitis disrupt the tight junction-associated biliary epithelial cell barrier.

BACKGROUND & AIMS: IgG4-related cholangitis is a chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis. A T-helper (Th) 2 cell cytokine profile predominates in liver tissues from these patients. We investigated whether Th2 cytokines disrupt the barrier function of biliary epithelial cells (BECs) in patients with IgG4-related cholangitis. METHODS: We assessed the Th2 cytokine profile in bile samples and brush cytology samples from 16 patients with IgG4-related cholangitis and respective controls, and evaluated transcription of tight junction (TJ)-associated proteins in primary BECs from these patients. The effect of Th2 cytokines on TJ-mediated BEC barrier function and wound closure was examined by immunoblot, transepithelial resistance, charge-selective Na(+)/Cl(-) permeability, and 4-kDa dextran flux analyses. RESULTS: Bile samples from patients with IgG4-related cholangitis had significant increases in levels of Th2 cytokines, interleukin (IL)-4, and IL-5. IL-13 was not detected in bile samples, but polymerase chain reaction analysis of whole-brush cytology samples from patients with IgG4-related cholangitis revealed increased levels of IL-13 mRNA, compared with controls. BECs isolated from the brush cytology samples revealed decreased levels of claudin-1 and increased levels of claudin-2 mRNAs. In vitro, IL-4 and IL-13 significantly reduced TJ-associated BEC barrier function by activating claudin-2-mediated paracellular pore pathways. Th2 cytokines also impaired wound closure in BEC monolayers. CONCLUSIONS: Th2 cytokines predominate in bile samples from patients with IgG4-related cholangitis and disrupt the TJ-mediated BEC barrier in vitro. Subsequent increases in biliary leaks might contribute to the pathogenesis of chronic biliary inflammation in these patients.

Toll-like receptor 4 variant D299G induces features of neoplastic progression in Caco-2 intestinal cells and is associated with advanced human colon cancer.

BACKGROUND & AIMS: The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions. METHODS: We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage. RESULTS: Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. CONCLUSIONS: TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.

LRRK2 is involved in the IFN-gamma response and host response to pathogens.

LRRK2 was previously identified as a defective gene in Parkinson's disease, and it is also located in a risk region for Crohn's disease. In this study, we aim to determine whether LRRK2 could be involved in immune responses. We show that LRRK2 expression is enriched in human immune cells. LRRK2 is an IFN-γ target gene, and its expression increased in intestinal tissues upon Crohn's disease inflammation. In inflamed intestinal tissues, LRRK2 is detected in the lamina propria macrophages, B-lymphocytes, and CD103-positive dendritic cells. Furthermore, LRRK2 expression enhances NF-κB-dependent transcription, suggesting its role in immune response signaling. Endogenous LRRK2 rapidly translocates near bacterial membranes, and knockdown of LRRK2 interferes with reactive oxygen species production during phagocytosis and bacterial killing. These observations indicate that LRRK2 is an IFN-γ target gene, and it might be involved in signaling pathways relevant to Crohn's disease pathogenesis.

Identification of Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated muramyl dipeptide transporters.

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappaB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappaB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.

Enhanced innate immune responsiveness and intolerance to intestinal endotoxins in human biliary epithelial cells contributes to chronic cholangitis.

BACKGROUND: Pattern recognition receptors (PRRs) orchestrate the innate immune defence in human biliary epithelial cells (BECs). Tight control of PRR signalling provides tolerance to physiological amounts of intestinal endotoxins in human bile to avoid constant innate immune activation in BECs. AIMS: We wanted to determine whether inappropriate innate immune responses to intestinal endotoxins contribute to the development and perpetuation of chronic biliary inflammation. METHODS: We examined PRR-mediated innate immune responses and protective endotoxin tolerance in primary BECs isolated from patients with primary sclerosing cholangitis (PSC), alcoholic liver disease and patients without chronic liver disease. Expression studies comprised northern blots, RT-PCR, Western blots and immunocytochemistry. Functional studies comprised immuno-precipitation Western blots, FACS for endotoxin uptake, and NF-κB activation assays and ELISA for secreted IL-8 and tumour necrosis factor (TNF)-α. RESULTS: Primary BECs from explanted PSC livers showed reversibly increased TLR and NOD protein expression and activation of the MyD88/IRAK signalling complex. Consecutively, PSC BECs exhibited inappropriate innate immune responses to endotoxins and did not develop immune tolerance after repeated endotoxin exposures. This endotoxin hyper-responsiveness was probably because of the stimulatory effect of abundantly expressed IFN-γ and TNF-α in PSC livers, which stimulated TLR4-mediated endotoxin signalling in BECs, leading to increased TLR4-mediated endotoxin incorporation and impaired inactivation of the TLR4 signalling cascade. As TNF-α inhibition partly restored protective innate immune tolerance, endogenous TNF-α secretion probably contributed to inappropriate endotoxin responses in BECs. CONCLUSION: Inappropriate innate immune responses to intestinal endotoxins and subsequent endotoxin intolerance because of enhanced PRR signalling in BECs probably contribute to chronic cholangitis.

A novel hybrid yeast-human network analysis reveals an essential role for FNBP1L in antibacterial autophagy.

Autophagy is a conserved cellular process required for the removal of defective organelles, protein aggregates, and intracellular pathogens. We used a network analysis strategy to identify novel human autophagy components based upon the yeast interactome centered on the core yeast autophagy proteins. This revealed the potential involvement of 14 novel mammalian genes in autophagy, several of which have known or predicted roles in membrane organization or dynamics. We selected one of these membrane interactors, FNBP1L (formin binding protein 1-like), an F-BAR-containing protein (also termed Toca-1), for further study based upon a predicted interaction with ATG3. We confirmed the FNBP1L/ATG3 interaction biochemically and mapped the FNBP1L domains responsible. Using a functional RNA interference approach, we determined that FNBP1L is essential for autophagy of the intracellular pathogen Salmonella enterica serovar Typhimurium and show that the autophagy process serves to restrict the growth of intracellular bacteria. However, FNBP1L appears dispensable for other forms of autophagy induced by serum starvation or rapamycin. We present a model where FNBP1L is essential for autophagy of intracellular pathogens and identify FNBP1L as a differentially used molecule in specific autophagic contexts. By using network biology to derive functional biological information, we demonstrate the utility of integrated genomics to novel molecule discovery in autophagy.

In vivo action of trefoil factor 2 (TFF2) to speed gastric repair is independent of cyclooxygenase.

OBJECTIVE: Trefoil factor (TFF) peptides are expressed in gastric tissues, where they are part of the epithelial defences. To complement previous in vitro work, the goal of the present study was to examine directly if TFF2 was essential for gastric restitution in vivo during the recovery from microscopic damage. DESIGN: TFF2 mutant (KO) mice were examined to study the epithelial repair process in vivo after laser-induced photodamage (LPD). Using two-photon laser energy absorption (710 nm), LPD was imposed on an approximately 3-5 cell region of surface epithelium in anaesthetised mouse stomach. Responses to damage were evaluated during confocal time-lapse microscopy; including area of damage and the extracellular pH adjacent to the damaged surface (Cl-NERF pH sensor). RESULTS: In control (TFF2+/+ and TFF2+/-) mice, damaged cells were exfoliated and the damaged epithelium was repaired by indomethacin. The resting surface pH was similar between control and TFF2-KO animals, but the post-LPD alkalisation of surface pH observed in control mice (pH 0.3 + or - 0.05, n=21) was attenuated in the TFF2-KO stomach (pH -0.08 + or - 0.09, n=18). Recobinant rat TFF3 partially rescued the attenuated surface pH change in TFF2-KO stomach, in the presence or absence of indomethacin. CONCLUSIONS: In the gastric epithelium in vivo, TFFs promote epithelial restitution via a mechanism that does not require cyclooxygenase activation. A novel role for TFFs to affect gastric surface pH is observed.

Colitis-associated variant of TLR2 causes impaired mucosal repair because of TFF3 deficiency.

BACKGROUND & AIMS: Goblet cells (GC) facilitate mucosal protection and epithelial barrier repair, yet the innate immune mechanisms that selectively drive GC functions have not been defined. The aim of this study was to determine whether Toll-like receptor (TLR) 2 and modulation of GC-derived trefoil factor (TFF) 3 are functionally linked in the intestine. METHODS: GC modulation was assessed using quantitative real-time polymerase chain reaction analysis (qRT-PCR), Western blotting, and confocal microscopy. Dextran sulfate sodium (DSS) colitis was induced in wild-type, TFF3(-/-), and TLR2(-/-) mice. Recombinant TLR2 ligand or TFF3 peptide were orally administered after DSS termination. Caco-2 cells overexpressing full-length TLR2 or mutant TLR2-R753Q were tested for TFF3 synthesis and functional-related effects in a wounding assay. RESULTS: Data from in vitro (Ls174T) and ex vivo models of murine and human GC reveal that TLR2 activation selectively induces synthesis of TFF3. In vivo studies using TFF3(-/-) or TLR2(-/-) mice demonstrate the ability for oral treatment with a TLR2 agonist to confer antiapoptotic protection of the intestinal mucosa against inflammatory stress-induced damage through TFF3. Recombinant TFF3 rescues TLR2-deficient mice from increased morbidity and mortality during acute colonic injury. Severe ulcerative colitis (UC) has recently been found to be associated with the R753Q polymorphism of the TLR2 gene. The relevance of the observed functional effect of TLR2 in regulating GC is confirmed by the finding that the UC-associated TLR2-R753Q variant is functionally deficient in the ability to induce TFF3 synthesis, thus leading to impaired wound healing. CONCLUSIONS: These data demonstrate a novel function of TLR2 in intestinal GC that links products of commensal bacteria to innate immune protection of the host via TFF3.

Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

Synthesis of biologically active biotinylated muramyl dipeptides.

Muramyl dipeptide (MDP) is believed to interact with an innate immune receptor, Nod2. To identify the cellular receptor for MDP, we have synthesized biotinylated MDP isomers and tested the ability of these compounds to activate Nod2 in a cell-based assay. We found that the modification of MDP does not perturb its ability to activate Nod2. These tagged versions of MDP will be useful to identify the cellular receptor of the immunostimulatory molecules.

The cytoskeletal scaffold Shank3 is recruited to pathogen-induced actin rearrangements.

The common gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC) and Salmonella typhimurium both reorganize the gut epithelial cell actin cytoskeleton to mediate pathogenesis, utilizing mimicry of the host signaling apparatus. The PDZ domain-containing protein Shank3, is a large cytoskeletal scaffold protein with known functions in neuronal morphology and synaptic signaling, and is also capable of acting as a scaffolding adaptor during Ret tyrosine kinase signaling in epithelial cells. Using immunofluorescent and functional RNA-interference approaches we show that Shank3 is present in both EPEC- and S. typhimurium-induced actin rearrangements and is required for optimal EPEC pedestal formation. We propose that Shank3 is one of a number of host synaptic proteins likely to play key roles in bacteria-host interactions.

TNF receptor type I-dependent activation of innate responses to reduce intestinal damage-associated mortality.

BACKGROUND & AIMS: Ligation of tumor necrosis factor (TNF) receptors (TNFRs) with TNF plays a critical role in the pathogenesis of human inflammatory bowel disease (IBD). However, it remains unclear which cell types activated through TNFR-associated signaling cascades are involved in the pathogenesis of colitis. METHODS: Recombination activating gene-1 (RAG) knockout (KO) (no T or B cells)-based TNFR double and triple KO mice were generated. Bone marrow (BM) chimera mice in which BM-derived myeloid cells, but not colonic epithelial cells (CECs), express TNFRs were also generated. Colitis was induced by administration of dextran sodium sulfate (DSS) in distilled water. Murine lines and chimeras were assessed for disease severity, histopathology, apoptotic cell rate, epithelial proliferation, and bacterial invasion rate. RESULTS: Following DSS administration, mice lacking both RAG and TNFR1 exhibited a high mortality (>80%) rate with an impaired CEC regeneration compared with RAG KO and RAG x TNFR2 double KO (DKO) mice. Transplantation of RAG KO-derived BM cells restored CEC regeneration and rescued the majority of recipient RAG x TNFR1 DKO mice from DSS-induced mortality. After BM transplantation, RAG x TNFR1 DKO mice exhibited an increased rate of apoptosis in the colonic lamina propria macrophages in association with the activation of caspases. In addition, BM reconstitution directly or indirectly enhanced the proliferation of CECs by activating mitogen-activated protein kinase and phosphoinositide-3 kinase/Akt pathways. CONCLUSIONS: TNFR1-signaling cascade in colonic myeloid lineage cells contributes to the suppression of acute damage-associated mortality presumably by controlling CEC homeostasis.