Role of Tumor Necrosis Factor Receptor 2 (TNFR2) in Colonic Epithelial Hyperplasia and Chronic Intestinal Inflammation in Mice

Gastroenterology. Jan, 2002  |  Pubmed ID: 11781288

Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammation-induced colonic epithelial alteration.

Decreased Bone Density in Splenectomized Gaucher Patients Receiving Enzyme Replacement Therapy

Blood Cells, Molecules & Diseases. Mar-Apr, 2002  |  Pubmed ID: 12064924

Little is known about the effect of enzyme replacement therapy (ERT) on the bone abnormalities in Gaucher disease. Splenectomized Gaucher patients tend to suffer the most severe skeletal complications. We hypothesized that vitamin D supplementation would act synergistically with glucocerebrosidase infusions to increase bone density in splenectomized Gaucher patients. In a 24-month study, 29 splenectomized Gaucher patients were randomized to three groups: Group 1, calcitriol (1,25-dihydroxyvitamin D3; 0.25-3.0 microg/day) alone for the first 6 months with the addition of ceredase/cerezyme at 60 IU/kg every 2 weeks during months 7-12; Group 2, calcitriol together with ceredase/cerezyme at 60 IU/kg every 2 weeks during months 1-6; and Group 3, enzyme only at 60 IU/kg body wt every 2 weeks. In all three groups, enzyme dose was halved after the first 6 months of therapy. The primary outcome measure was bone mineral density of the lumbar spine measured by single-energy quantitative CT. Bone density by single-energy CT (P = 0.001) and by dual-energy CT (P = 0.06) declined overall, but there was no significant difference between the groups. Calcitriol had no significant effect on bone density. Fat fraction in lumbar spine increased (P = 0.000) and skeletal MRI scores improved. Bone-specific alkaline phosphatase (P = 0.002) and serum osteocalcin increased (P = 0.008), while blood cyclic AMP and urinary deoxypyridinoline did not change appreciably. Hemoglobin, platelet counts, and liver volume significantly improved. We conclude that ERT alone, or in combination with calcitriol, cannot repair the bone composition in splenectomized adult Gaucher patients. Alternatively, measuring trabecular bone density may be an inadequate marker of clinical efficacy for treating skeletal involvement in Gaucher disease.

Transforming Growth Factor-beta Mediates Intestinal Healing and Susceptibility to Injury in Vitro and in Vivo Through Epithelial Cells

The American Journal of Pathology. Feb, 2003  |  Pubmed ID: 12547717

In vitro studies suggest that transforming growth factor (TGF)-beta has potent effects on gastrointestinal mucosal integrity, wound repair, and neoplasia. However, the multiplicity of actions of this peptide on many different cell types confounds efforts to define the role of TGF-beta within the intestinal epithelium in vivo. To delineate these effects selective blockade of intestinal epithelial TGF-beta activity was undertaken through targeted expression of a dominant-negative (DN) TGF-beta RII to intestinal epithelial cells in vitro and in vivo. Stable intestinal epithelial cell (IEC)-6 lines overexpressing TGF-beta RII-DN (nucleotides -7 to 573) were established. Transgenic mice overexpressing TGF-beta RII-DN under the regulation of a modified liver fatty acid-binding promoter (LFABP-PTS4) were constructed. In vitro healing was assessed by wounding of confluent monolayers. Colitis was induced by the addition of dextran sodium sulfate (2.5 to 7.5% w/v) to their drinking water. Overexpression of TGF-beta RII-DN in intestinal epithelial cell-6 cells resulted in a marked reduction in cell migration and TGF-beta-stimulated wound healing in vitro. TGF-beta RII-DN transgenic mice did not exhibit baseline intestinal inflammation or changes in survival, body weight, epithelial cell proliferation, aberrant crypt foci, or tumor formation. TGF-beta RII-DN mice were markedly more susceptible to dextran sodium sulfate-induced colitis and exhibited impaired recovery after colonic injury. TGF-beta is required for intestinal mucosal healing and TGF-beta modulation of the intestinal epithelium plays a central role in determining susceptibility to injury.

Colonic Epithelial Functional Phenotype Varies with Type and Phase of Experimental Colitis

Gastroenterology. Jul, 2003  |  Pubmed ID: 12851880

Colonic crypt elongation occurs during both chronic colitis and in the recovery phase of acute colitis. The impact of these alterations on epithelial cell functions is not fully defined.

The Scaffold Protein CNK1 Interacts with the Tumor Suppressor RASSF1A and Augments RASSF1A-induced Cell Death

The Journal of Biological Chemistry. Jul, 2004  |  Pubmed ID: 15075335

The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. Herein we demonstrate a novel aspect of CNK1 function, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.

Bioluminescence Resonance Energy Transfer Identify Scaffold Protein CNK1 Interactions in Intact Cells

FEBS Letters. Jan, 2005  |  Pubmed ID: 15670823

Connector enhancer of KSR (CNK) proteins have been proposed to act as scaffolds in the Ras-MAPK pathway. In this work, using in vivo bioluminescence resonance energy transfer (BRET) assays and in vitro co-immunoprecipitation, we show that hCNK1 interacts with the active form of Rho A (G14V) proteins. The domain of hCNK1 that allows binding to Rho proteins involves the C-terminal PH domain. Overexpression of hCNK1 does not affect the actin cytoskeleton and does not modify the appearance of stress fibers in cells overexpressing a constitutively active form of RhoA. In contrast, hCNK1 was able to significantly decrease the RhoA-induced transcriptional activity of the serum response element (SRE) without effect on the Ras-induced SRE activation. These results identify hCNK1 as a specific partner of Rho proteins both in vitro and in vivo and suggest a role of hCNK1 in the signal transduction of Rho proteins.

Ca2+/calmodulin-dependent Protein Kinase II is a Modulator of CARMA1-mediated NF-kappaB Activation

Molecular and Cellular Biology. Jul, 2006  |  Pubmed ID: 16809782

CARMA1 is a central regulator of NF-kappaB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-kappaB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca(2+)/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-kappaB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-kappaB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-kappaB activation.

Discs-large Homolog 1 Regulates Smooth Muscle Orientation in the Mouse Ureter

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2006  |  Pubmed ID: 17172448

Discs-large homolog 1 (DLGH1) is a mouse ortholog of the Drosophila discs-large (DLG) tumor suppressor protein, a founding member of the PDZ and MAGUK protein families. DLG proteins play important roles in regulating cell proliferation, epithelial cell polarity, and synapse formation and function. Here, we generated a null allele of Dlgh1 and studied its role in urogenital development. Dlgh1(-/-) mice developed severe urinary tract abnormalities, including congenital hydronephrosis, which is the leading cause of renal failure in infants and children. DLGH1 is expressed in the developing ureter; in its absence, the stromal cells that normally lie between the urothelial and smooth muscle layers were missing. Moreover, in ureteric smooth muscle, the circular smooth muscle cells were misaligned in a longitudinal orientation. These abnormalities in the ureter led to severely impaired ureteric peristalsis. Similar smooth muscle defects are observed frequently in patients with ureteropelvic junction obstruction, a common form of hydronephrosis. Our results suggest that (i) besides its well documented role in regulating epithelial polarity, Dlgh1 also regulates smooth muscle orientation, and (ii) human DLG1 mutations may contribute to hereditary forms of hydronephrosis.

Genome-wide Association Study Identifies New Susceptibility Loci for Crohn Disease and Implicates Autophagy in Disease Pathogenesis

Nature Genetics. May, 2007  |  Pubmed ID: 17435756

We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10(-10)) with an intergenic region on 10q21.1 and a coding variant in ATG16L1, the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B, NCF4 and a predicted gene on 16q24.1 (FAM92B). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.

Molecular Pathogenesis of Inflammatory Bowel Disease: Genotypes, Phenotypes and Personalized Medicine

Annals of Medicine. 2007  |  Pubmed ID: 17457716

Crohn's disease (CD) and ulcerative colitis (UC), also known as inflammatory bowel diseases (IBD), are characterized by chronic inflammation of the gastrointestinal tract. IBD is among the few complex diseases for which several genomic regions and specific genes have been identified and confirmed in multiple replication studies. We will review the different loci implicated in disease risk in the context of three proposed mechanisms leading to chronic inflammation of the gut mucosa: 1) deregulation of the innate immune response to enteric microflora or pathogens; 2) increased permeability across the epithelial barrier; and 3) defective regulation of the adaptive immune system. As our knowledge of genetic variation, analytical approaches and technology improves, additional genetic risk factors are expected to be identified. With the identification of novel risk variants, additional pathophysiological mechanisms are likely to emerge. The resulting discoveries will further our molecular understanding of IBD, potentially leading to improved disease classification and rational drug design. Moreover, these approaches and tools can be applied in the context of variable drug response with the goal of providing more personalized clinical management of patients with IBD.

AKAP13, a RhoA GTPase-specific Guanine Exchange Factor, is a Novel Regulator of TLR2 Signaling

The Journal of Biological Chemistry. Nov, 2007  |  Pubmed ID: 17878165

Members of the guanine exchange factor (GEF) family of scaffold proteins are involved in the integration of signal flow downstream of many receptors in adaptive immunity. However, the full complement of GEFs that function downstream of Toll-like receptors (TLRs) requires further identification and functional understanding. By systematically integrating expression profiles from immune and epithelial cells with functional studies, we demonstrate that protein kinase A anchoring protein 13 (AKAP13), a scaffold protein with GEF activity, is an activator of NF-kappaB downstream of TLR2 signaling. Stimulation of the human macrophage cell line THP-1 and epithelial cells with a TLR2 ligand caused a significant up-regulation in AKAP13 mRNA, corresponding to an increase in protein expression. Analysis of TLR2 reporter cell lines deficient in AKAP13 expression revealed significantly reduced NF-kappaB activation and reduced secretion of interleukin-8 and MCP-1 in response to specific ligand stimulation. Furthermore, NF-kappaB activation was partially inhibited by a GEF-deficient AKAP13 mutant. AKAP13 was also involved in phosphorylation of JNK but not of extracellular signal-regulated kinase ERK1 and -2 following ligand stimulation. Together, our results suggest that AKAP13 plays a role in TLR2-mediated NF-kappaB activation and suggest that GEF-containing scaffold proteins may confer specificity to innate immune responses downstream of TLRs.

DLG1 is an Anchor for the E3 Ligase MARCH2 at Sites of Cell-cell Contact

Cellular Signalling. Jan, 2008  |  Pubmed ID: 17980554

PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.

IL-22 Ameliorates Intestinal Inflammation in a Mouse Model of Ulcerative Colitis

The Journal of Clinical Investigation. Feb, 2008  |  Pubmed ID: 18172556

Expression of IL-22 is induced in several human inflammatory conditions, including inflammatory bowel disease (IBD). Expression of the IL-22 receptor is restricted to innate immune cells; however, the role of IL-22 in colitis has not yet been defined. We developed what we believe to be a novel microinjection-based local gene-delivery system that is capable of targeting the inflamed intestine. Using this approach, we demonstrated a therapeutic potency for IL-22-mediated activation of the innate immune pathway in a mouse model of Th2-mediated colitis that induces disease with characteristics similar to that of IBD ulcerative colitis (UC). IL-22 gene delivery enhanced STAT3 activation specifically within colonic epithelial cells and induced both STAT3-dependent expression of mucus-associated molecules and restitution of mucus-producing goblet cells. Importantly, IL-22 gene delivery led to rapid amelioration of local intestinal inflammation. The amelioration of disease by IL-22 was mediated by enhanced mucus production. In addition, local gene delivery was used to inhibit IL-22 activity through overexpression of IL-22-binding protein. Treatment with IL-22-binding protein suppressed goblet cell restitution during the recovery phase of a dextran sulfate sodium-induced model of acute colitis. These data demonstrate what we believe to be a novel function for IL-22 in the intestine and suggest the potency of a local IL-22 gene-delivery system for treating UC.

Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

Science (New York, N.Y.). Feb, 2008  |  Pubmed ID: 18187620

HIV-1 exploits multiple host proteins during infection. We performed a large-scale small interfering RNA screen to identify host factors required by HIV-1 and identified more than 250 HIV-dependency factors (HDFs). These proteins participate in a broad array of cellular functions and implicate new pathways in the viral life cycle. Further analysis revealed previously unknown roles for retrograde Golgi transport proteins (Rab6 and Vps53) in viral entry, a karyopherin (TNPO3) in viral integration, and the Mediator complex (Med28) in viral transcription. Transcriptional analysis revealed that HDF genes were enriched for high expression in immune cells, suggesting that viruses evolve in host cells that optimally perform the functions required for their life cycle. This effort illustrates the power with which RNA interference and forward genetics can be used to expose the dependencies of human pathogens such as HIV, and in so doing identify potential targets for therapy.

Defective Signal Transduction in B Lymphocytes Lacking Presenilin Proteins

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2008  |  Pubmed ID: 18195359

The mammalian presenilin (PS) proteins mediate the posttranslational cleavage of several protein substrates, including amyloid precursor protein, Notch family members, and CD44, but they have also been suggested to function in diverse cellular processes, including calcium-dependent signaling and apoptosis. We carried out an integrative computational study of multiple genomic datasets, including RNA expression, protein interaction, and pathway analyses, which implicated PS proteins in Toll-like receptor signaling. To test these computational predictions, we analyzed mice carrying a conditional allele of PS1 and a germ line-inactivating allele of PS2, together with Cre site-specific recombinase expression under the influence of CD19 control sequences. Notably, B cells deficient in both PS1 and PS2 function have an unexpected and substantial deficit in both lipopolysaccharide and B cell antigen receptor-induced proliferation and signal transduction events, including a defect in anti-IgM-mediated calcium flux. Taken together, these results demonstrate a fundamental and unanticipated role for PS proteins in B cell function and emphasize the potency of (systems level) integrative analysis of whole-genome datasets in identifying novel biologic signal transduction relationships. Our findings also suggest that pharmacologic inhibition of PS for the treatment of conditions such as Alzheimer's disease may have potential consequences for immune system function.

Autophagy As an Important Process in Gut Homeostasis and Crohn's Disease Pathogenesis

Gut. Jun, 2008  |  Pubmed ID: 18272545

Defining Molecular Classifications and Targets in Gastroenteropancreatic Neuroendocrine Tumors Through DNA Microarray Analysis

Endocrine-related Cancer. Mar, 2008  |  Pubmed ID: 18310291

Current classifications of human gastroenteropancreatic neuroendocrine tumors (NETs) are inconsistent and based upon histopathologic but not molecular features. We sought to compare a molecular classification with the World Health Organization (WHO) histologic classification, identify genes that may be important for tumor progression, and determine whether gastrointestinal NETs (GI-NETs) differ in their molecular profile from pancreatic NETs (PNETs). DNA microarray analysis was performed to identify differentially expressed genes in PNETs and GI-NETs. Confirmation of expression levels was obtained by quantitative real-time PCR. Immunoblotting and mutational analysis were performed for selected genes. Hierarchical clustering of 19 PNETs revealed a 'benign' and 'malignant' cluster that corresponded well with the WHO categories of well-differentiated endocrine tumor (WDET) and well-differentiated endocrine carcinoma (WDEC) respectively. FEV, adenylate cyclase 2 (ADCY2), nuclear receptor subfamily 4, group A, member 2 (NR4A2), and growth arrest and DNA-damage-inducible, beta (GADD45b) were the most highly up-regulated genes in the malignant group of PNETs. Platelet-derived growth factor receptor (PDGFR) was expressed in both WDETs and WDECs, and phosphorylation of PDGFR-beta was observed in 83% of all PNETs. Malignant ileal GI-NETs exhibited a distinctive gene expression profile, and extracellular matrix protein 1 (ECM), vesicular monoamine member 1 (VMAT1), galectin 4 (LGALS4), and RET Proto-oncogene (RET) were highly up-regulated genes. Gene expression profiles reflect the current WHO classification and can distinguish benign from malignant PNETs and also PNETs from GI-NETs. This suggests that molecular profiling may enhance tumor classification schemes. Potential gene targets have also been identified, and PDGFR and RET are candidates that may represent novel therapeutic targets.

SPTLC1 Binds ABCA1 to Negatively Regulate Trafficking and Cholesterol Efflux Activity of the Transporter

Biochemistry. Jun, 2008  |  Pubmed ID: 18484747

ABCA1 transport of cholesterol and phospholipids to nascent HDL particles plays a central role in lipoprotein metabolism and macrophage cholesterol homeostasis. ABCA1 activity is regulated both at the transcriptional level and at the post-translational level. To explore mechanisms involved in the post-translational regulation of the transporter, we have used affinity purification and mass spectrometry to identify proteins that bind ABCA1 and influence its activity. Previously, we demonstrated that an interaction between beta1-syntrophin stimulated ABCA1 activity, at least in part, be slowing the degradation of the transporter. This work demonstrates that one subunit of the serine palmitoyltransferase enzyme, SPTLC1, but not subunit 2 (SPTLC2), is copurified with ABCA1 and negatively regulates its function. In human THP-I macrophages and in mouse liver, the ABCA1-SPTLC1 complex was detected by co-immunoprecipitation, demonstrating that the interaction occurs in cellular settings where ABCA1 activity is critical for HDL genesis. Pharmacologic inhibition of SPTLC1 with myriocin, which resulted in the disruption of the SPTLC1-ABCA1 complex, and siRNA knockdown of SPTLC1 expression both stimulated ABCA1 efflux by nearly 60% ( p < 0.05). In contrast, dominant-negative mutants of SPTLC1 inhibited ABCA1 efflux, indicating that a reduced level of sphingomyelin synthesis could not explain the effect of myriocin on ABCA1 activity. In 293 cells, the SPTLC1 inhibition of ABCA1 activity led to the blockade of the exit of ABCA1 from the endoplasmic reticulum. In contrast, myriocin treatment of macrophages increased the level of cell surface ABCA1. In composite, these results indicate that the physical interaction of ABCA1 and SPTLC1 results in reduction of ABCA1 activity and that inhibition of this interaction produces enhanced cholesterol efflux.

Neither Hide nor Hair: the Difficulty of Identifying Useful Disease Biomarkers

Gastroenterology. Jun, 2008  |  Pubmed ID: 18486617

Genome-wide Association Defines More Than 30 Distinct Susceptibility Loci for Crohn's Disease

Nature Genetics. Aug, 2008  |  Pubmed ID: 18587394

Several risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further, we combined data from three studies on Crohn's disease (a total of 3,230 cases and 4,829 controls) and carried out replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 additional loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1 and ITLN1. The expanded molecular understanding of the basis of this disease offers promise for informed therapeutic development.

Genome-wide Association Studies: a New Window into Immune-mediated Diseases

Nature Reviews. Immunology. Aug, 2008  |  Pubmed ID: 18654571

Given the recent explosion of genetic discoveries, 2007 is becoming known to human geneticists as the year of genome-wide association studies. In fact, more genetic risk factors for common diseases were identified in this one year than had been collectively reported before 2007. In particular, 2007 witnessed the discovery of many genes that influence susceptibility to individual immune-mediated diseases, as well as other genes that are associated with susceptibility to more than one disease. Although much work remains to be done, in this Review we discuss what effect these studies are having on our understanding of disease pathogenesis and their potential impact on future immunology studies.

Hoxc6 is Overexpressed in Gastrointestinal Carcinoids and Interacts with JunD to Regulate Tumor Growth

Gastroenterology. Sep, 2008  |  Pubmed ID: 18655788

The molecular alterations that underlie carcinoid tumor pathogenesis remain poorly defined. The homeobox gene HOXC6 was highly up-regulated in human gastrointestinal carcinoid tumors, and we sought to define its pathogenic role.

RNA Interference Screen for Human Genes Associated with West Nile Virus Infection

Nature. Sep, 2008  |  Pubmed ID: 18690214

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

A Key Role for Autophagy and the Autophagy Gene Atg16l1 in Mouse and Human Intestinal Paneth Cells

Nature. Nov, 2008  |  Pubmed ID: 18849966

Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.

Impaired Autophagy of an Intracellular Pathogen Induced by a Crohn's Disease Associated ATG16L1 Variant

PloS One. 2008  |  Pubmed ID: 18852889

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.

Role for Beta-catenin and HOX Transcription Factors in Caenorhabditis Elegans and Mammalian Host Epithelial-pathogen Interactions

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2008  |  Pubmed ID: 18981407

We used the model nematode Caenorhabditis elegans infected with the human pathogen Staphylococcus aureus to identify components of epithelial immunity. Transcriptional profiling and reverse genetic analysis revealed that mutation of the C. elegans beta-catenin homolog bar-1 or the downstream homeobox gene egl-5 results in a defective response and hypersensitivity to S. aureus infection. Epistasis analysis showed that bar-1 and egl-5 function in parallel to previously described C. elegans immune-response pathways. Overexpression of human homologs of egl-5 modulated NF-kappaB-dependent TLR2 signaling in epithelial cells. These data suggest that beta-catenin and homeobox genes play an important and conserved role in innate immune defense.

Deletion Polymorphism Upstream of IRGM Associated with Altered IRGM Expression and Crohn's Disease

Nature Genetics. Sep, 2008  |  Pubmed ID: 19165925

Following recent success in genome-wide association studies, a critical focus of human genetics is to understand how genetic variation at implicated loci influences cellular and disease processes. Crohn's disease (CD) is associated with SNPs around IRGM, but coding-sequence variation has been excluded as a source of this association. We identified a common, 20-kb deletion polymorphism, immediately upstream of IRGM and in perfect linkage disequilibrium (r2 = 1.0) with the most strongly CD-associated SNP, that causes IRGM to segregate in the population with two distinct upstream sequences. The deletion (CD risk) and reference (CD protective) haplotypes of IRGM showed distinct expression patterns. Manipulation of IRGM expression levels modulated cellular autophagy of internalized bacteria, a process implicated in CD. These results suggest that the CD association at IRGM arises from an alteration in IRGM regulation that affects the efficacy of autophagy and identify a common deletion polymorphism as a likely causal variant.

Identification of a Molecular Signaling Network That Regulates a Cellular Necrotic Cell Death Pathway

Cell. Dec, 2008  |  Pubmed ID: 19109899

Stimulation of death receptors by agonists such as FasL and TNFalpha activates apoptotic cell death in apoptotic-competent conditions or a type of necrotic cell death dependent on RIP1 kinase, termed necroptosis, in apoptotic-deficient conditions. In a genome-wide siRNA screen for regulators of necroptosis, we identify a set of 432 genes that regulate necroptosis, a subset of 32 genes that act downstream and/or as regulators of RIP1 kinase, 32 genes required for death-receptor-mediated apoptosis, and 7 genes involved in both necroptosis and apoptosis. We show that the expression of subsets of the 432 genes is enriched in the immune and nervous systems, and cellular sensitivity to necroptosis is regulated by an extensive signaling network mediating innate immunity. Interestingly, Bmf, a BH3-only Bcl-2 family member, is required for death-receptor-induced necroptosis. Our study defines a cellular signaling network that regulates necroptosis and the molecular bifurcation that controls apoptosis and necroptosis.

Differential Requirement for CARMA1 in Agonist-selected T-cell Development

European Journal of Immunology. Jan, 2009  |  Pubmed ID: 19130560

Caspase recruitment domain-containing membrane-associated guanylate kinase protein-1 (CARMA1) is a critical component of the NF-kappaB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist-selected T-cell subsets such as Treg, NKT cells, and CD8-alpha alpha T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1-deficient mice (CARMA1(-/-)) have altered populations of specific subsets of agonist-selected T cells. Specifically, CARMA1(-/-) mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild-type mice. Interestingly, CD8-alpha alpha T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1(-/-) mice, whereas memory-phenotype CD4(+) T cells (CD62L(low)/CD44(high)) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist-selected T-cell subsets. Overall, these data reveal an important but differential role for CARMA1-mediated TCR signaling in T-cell development.

A Genome-wide Genetic Screen for Host Factors Required for Hepatitis C Virus Propagation

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2009  |  Pubmed ID: 19717417

Hepatitis C virus (HCV) infection is a major cause of end-stage liver disease and a leading indication for liver transplantation. Current therapy fails in many instances and is associated with significant side effects. HCV encodes only a few proteins and depends heavily on host factors for propagation. Each of these host dependencies is a potential therapeutic target. To find host factors required by HCV, we completed a genome-wide small interfering RNA (siRNA) screen using an infectious HCV cell culture system. We applied a two-part screening protocol to allow identification of host factors involved in the complete viral lifecycle. The candidate genes found included known or previously identified factors, and also implicate many additional host cell proteins in HCV infection. To create a more comprehensive view of HCV and host cell interactions, we performed a bioinformatic meta-analysis that integrates our data with those of previous functional and proteomic studies. The identification of host factors participating in the complete HCV lifecycle will both advance our understanding of HCV pathogenesis and illuminate therapeutic targets.

A Functional Genomic Screen Identifies Cellular Cofactors of Hepatitis C Virus Replication

Cell Host & Microbe. Mar, 2009  |  Pubmed ID: 19286138

Hepatitis C virus (HCV) chronically infects 3% of the world's population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies.

A Novel Hybrid Yeast-human Network Analysis Reveals an Essential Role for FNBP1L in Antibacterial Autophagy

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2009  |  Pubmed ID: 19342671

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.

The Cytoskeletal Scaffold Shank3 is Recruited to Pathogen-induced Actin Rearrangements

Experimental Cell Research. Jul, 2009  |  Pubmed ID: 19371741

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.

An Integrative Genomics Approach Identifies Hypoxia Inducible Factor-1 (HIF-1)-target Genes That Form the Core Response to Hypoxia

Nucleic Acids Research. Aug, 2009  |  Pubmed ID: 19491311

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a central role in the transcriptional response to oxygen flux. To gain insight into the molecular pathways regulated by HIF-1, it is essential to identify the downstream-target genes. We report here a strategy to identify HIF-1-target genes based on an integrative genomic approach combining computational strategies and experimental validation. To identify HIF-1-target genes microarrays data sets were used to rank genes based on their differential response to hypoxia. The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites. Genes were scored and ranked based on their response to hypoxia and their HIF-binding site score. Using this strategy we recovered 41% of the previously confirmed HIF-1-target genes that responded to hypoxia in the microarrays and provide a catalogue of predicted HIF-1 targets. We present experimental validation for ANKRD37 as a novel HIF-1-target gene. Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets.

Identifying Relationships Among Genomic Disease Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions

PLoS Genetics. Jun, 2009  |  Pubmed ID: 19557189

Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn's disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions--that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/).

On the Level: IRGM Gene Function is All About Expression

Autophagy. Jan, 2009  |  Pubmed ID: 19029815

Crohn disease is a complex, multigenic, chronic inflammatory bowel disease of uncertain etiology. Recent advances in genetics, including high-throughput single-nucleotide polymorphism typing platforms and deep sequencing technologies have begun to shed light upon disease predisposition and pathogenesis. Autophagy is emerging as a key player in both innate and adaptive immunity, as well as tissue homeostasis and development in the gut. Here we describe our recent studies into the Crohn disease-associated Immunity-Related GTPase family, M (IRGM) gene and our discovery of a large risk-conferring upstream deletion. We discuss the effects of this deletion upon expression levels of IRGM alleles and how tissue-specific expression might be affected by the promoter polymorphism. In addition, we comment upon the potential roles of IRGM in autophagy of intracellular pathogens, and the challenges ahead for further elucidating IRGM function.

HIF-1alpha and HIF-2alpha Have Divergent Roles in Colon Cancer

International Journal of Cancer. Journal International Du Cancer. Feb, 2009  |  Pubmed ID: 19030186

Hypoxia-inducible factor (HIF)-1 and HIF-2 are heterodimeric transcription factors that mediate the cellular response to hypoxia. Their key regulatory subunits, HIF-1alpha and HIF-2alpha, are induced similarly by hypoxia, but their functional roles in cancer may be distinct and isoform-specific. SW480 colon cancer cells with stable expression of siRNA to HIF-1alpha or HIF-2alpha or both were established. HIF-1alpha-deficient cells displayed lower rates of proliferation and migration, but HIF-2alpha-deficient cells exhibited enhanced anchorage independent growth in a soft agar assay. Xenograft studies revealed that HIF-1alpha deficiency inhibited overall tumor growth, whereas deficiency of HIF-2alpha stimulated tumor growth. In human colon cancer tissues, expression of HIF-1alpha and to a lesser extent, HIF-2alpha, was linked to upregulation of VEGF and tumor angiogenesis. However, loss of expression of HIF-2alpha but not HIF-1alpha was strongly correlated with advanced tumor stage. DNA microarray analysis identified distinct sets of HIF-1alpha and HIF-2alpha target genes that may explain these phenotypic differences. Collectively, these findings suggest that HIF isoforms may have differing cellular functions in colon cancer. In particular, HIF-1alpha promoted the growth of SW480 colon cancer cells but HIF-2alpha appeared to restrain growth. Consequently, therapeutic approaches that target HIF may need to consider these isoform-specific properties.

Autophagy, Immunity and Human Disease

Current Opinion in Gastroenterology. Nov, 2009  |  Pubmed ID: 19826372

To give an overview of autophagy and its effects on innate and adaptive immunity and touch on some of the roles of autophagy in disease.

Regulation of Inflammatory Responses by Gut Microbiota and Chemoattractant Receptor GPR43

Nature. Oct, 2009  |  Pubmed ID: 19865172

The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in 'healthy' microbiota such as Bifidobacterium and Bacteriodes, and a concurrent reduction in SCFAs. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2), and here we show that SCFA-GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43(-/-)) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43(-/-) immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

Common Variants at Five New Loci Associated with Early-onset Inflammatory Bowel Disease

Nature Genetics. Dec, 2009  |  Pubmed ID: 19915574

The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are common causes of morbidity in children and young adults in the western world. Here we report the results of a genome-wide association study in early-onset IBD involving 3,426 affected individuals and 11,963 genetically matched controls recruited through international collaborations in Europe and North America, thereby extending the results from a previous study of 1,011 individuals with early-onset IBD. We have identified five new regions associated with early-onset IBD susceptibility, including 16p11 near the cytokine gene IL27 (rs8049439, P = 2.41 x 10(-9)), 22q12 (rs2412973, P = 1.55 x 10(-9)), 10q22 (rs1250550, P = 5.63 x 10(-9)), 2q37 (rs4676410, P = 3.64 x 10(-8)) and 19q13.11 (rs10500264, P = 4.26 x 10(-10)). Our scan also detected associations at 23 of 32 loci previously implicated in adult-onset Crohn's disease and at 8 of 17 loci implicated in adult-onset ulcerative colitis, highlighting the close pathogenetic relationship between early- and adult-onset IBD.

The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus

Cell. Dec, 2009  |  Pubmed ID: 20064371

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferon's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens.

CARMA3 Mediates Lysophosphatidic Acid-stimulated Cytokine Secretion by Bronchial Epithelial Cells

American Journal of Respiratory Cell and Molecular Biology. Mar, 2009  |  Pubmed ID: 18757306

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.

Genome-wide Association Identifies Multiple Ulcerative Colitis Susceptibility Loci

Nature Genetics. Apr, 2010  |  Pubmed ID: 20228799

Ulcerative colitis is a chronic, relapsing inflammatory condition of the gastrointestinal tract with a complex genetic and environmental etiology. In an effort to identify genetic variation underlying ulcerative colitis risk, we present two distinct genome-wide association studies of ulcerative colitis and their joint analysis with a previously published scan, comprising, in aggregate, 2,693 individuals with ulcerative colitis and 6,791 control subjects. Fifty-nine SNPs from 14 independent loci attained an association significance of P < 10(-5). Seven of these loci exceeded genome-wide significance (P < 5 x 10(-8)). After testing an independent cohort of 2,009 cases of ulcerative colitis and 1,580 controls, we identified 13 loci that were significantly associated with ulcerative colitis (P < 5 x 10(-8)), including the immunoglobulin receptor gene FCGR2A, 5p15, 2p16 and ORMDL3 (orosomucoid1-like 3). We confirmed association with 14 previously identified ulcerative colitis susceptibility loci, and an analysis of acknowledged Crohn's disease loci showed that roughly half of the known Crohn's disease associations are shared with ulcerative colitis. These data implicate approximately 30 loci in ulcerative colitis, thereby providing insight into disease pathogenesis.

Gene Enrichment Profiles Reveal T-cell Development, Differentiation, and Lineage-specific Transcription Factors Including ZBTB25 As a Novel NF-AT Repressor

Blood. Jul, 2010  |  Pubmed ID: 20410506

The identification of transcriptional regulatory networks, which control tissue-specific development and function, is of central importance to the understanding of lymphocyte biology. To decipher transcriptional networks in T-cell development and differentiation we developed a browsable expression atlas and applied a novel quantitative method to define gene sets most specific to each of the represented cell subsets and tissues. Using this system, body atlas size datasets can be used to examine gene enrichment profiles from a cell/tissue perspective rather than gene perspective, thereby identifying highly enriched genes within a cell type, which are often key to cellular differentiation and function. A systems analysis of transcriptional regulators within T cells during different phases of development and differentiation resulted in the identification of known key regulators and uncharacterized coexpressed regulators. ZBTB25, a BTB-POZ family transcription factor, was identified as a highly T cell-enriched transcription factor. We provide evidence that ZBTB25 functions as a negative regulator of nuclear factor of activated T cells (NF-AT) activation, such that RNA interference mediated knockdown resulted in enhanced activation of target genes. Together, these findings suggest a novel mechanism for NF-AT mediated gene expression and the compendium of expression data provides a quantitative platform to drive exploration of gene expression across a wide range of cell/tissue types.

Characterization and Analysis of Human Chordoma Cell Lines

Spine. Jun, 2010  |  Pubmed ID: 20461036

An experimental study to investigate the characterization of 3 chordoma cell lines.

A Systems Biology Viewpoint on Autophagy in Health and Disease

Current Opinion in Gastroenterology. Jul, 2010  |  Pubmed ID: 20571384

The field of autophagy is rapidly expanding to encompass many important areas of cell biology, physiology and disease. Recent discoveries and tools allow the connection of the autophagy pathway to other cellular signals and processes, thus beginning a systematic approach to elucidation of autophagy components, functions and connections.

Autophagy at the Gut Interface: Mucosal Responses to Stress and the Consequences for Inflammatory Bowel Diseases

Inflammatory Bowel Diseases. Jan, 2010  |  Pubmed ID: 19575363

Autophagy is a conserved homeostatic process by which cells degrade and recycle cytoplasmic contents and organelles. Recently, autophagy has come to prominence as a factor in many disease states, including inflammatory bowel diseases. In this review we explore the recent discoveries in autophagy and how these relate to the special conditions experienced by the gut mucosa. We will pay particular attention to autophagy as an innate immune process and its role in the development and education of the adaptive immune system.

Crohn's Disease-associated Adherent-invasive E. Coli Are Selectively Favoured by Impaired Autophagy to Replicate Intracellularly

Cellular Microbiology. Jan, 2010  |  Pubmed ID: 19747213

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.

A Genome-wide SiRNA Screen Reveals Multiple MTORC1 Independent Signaling Pathways Regulating Autophagy Under Normal Nutritional Conditions

Developmental Cell. Jun, 2010  |  Pubmed ID: 20627085

Autophagy is a cellular catabolic mechanism that plays an essential function in protecting multicellular eukaryotes from neurodegeneration, cancer, and other diseases. However, we still know very little about mechanisms regulating autophagy under normal homeostatic conditions when nutrients are not limiting. In a genome-wide human siRNA screen, we demonstrate that under normal nutrient conditions upregulation of autophagy requires the type III PI3 kinase, but not inhibition of mTORC1, the essential negative regulator of starvation-induced autophagy. We show that a group of growth factors and cytokines inhibit the type III PI3 kinase through multiple pathways, including the MAPK-ERK1/2, Stat3, Akt/Foxo3, and CXCR4/GPCR, which are all known to positively regulate cell growth and proliferation. Our study suggests that the type III PI3 kinase integrates diverse signals to regulate cellular levels of autophagy, and that autophagy and cell proliferation may represent two alternative cell fates that are regulated in a mutually exclusive manner.

Wnt Signaling Regulates Mitochondrial Physiology and Insulin Sensitivity

Genes & Development. Jul, 2010  |  Pubmed ID: 20634317

Mitochondria serve a critical role in physiology and disease. The genetic basis of mitochondrial regulation in mammalian cells has not yet been detailed. We performed a large-scale RNAi screen to systematically identify genes that affect mitochondrial abundance and function. This screen revealed previously unrecognized roles for >150 proteins in mitochondrial regulation. We report that increased Wnt signals are a potent activator of mitochondrial biogenesis and reactive oxygen species (ROS) generation, leading to DNA damage and acceleration of cellular senescence in primary cells. The signaling protein insulin receptor substrate-1 (IRS-1), shown here to be a transcriptional target of Wnt, is induced in this setting. The increased level of IRS-1 drives activation of mitochondrial biogenesis; furthermore, in insulin-responsive cell types, it enhances insulin signaling, raising the possibility that Wnt proteins may be used to modulate glucose homeostasis. Our results identify a key component of the mitochondrial regulatory apparatus with a potentially important link to metabolic and degenerative disorders.

Genome-wide Analysis Reveals Mechanisms Modulating Autophagy in Normal Brain Aging and in Alzheimer's Disease

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2010  |  Pubmed ID: 20660724

Dysregulation of autophagy, a cellular catabolic mechanism essential for degradation of misfolded proteins, has been implicated in multiple neurodegenerative diseases. However, the mechanisms that lead to the autophagy dysfunction are still not clear. Based on the results of a genome-wide screen, we show that reactive oxygen species (ROS) serve as common mediators upstream of the activation of the type III PI3 kinase, which is critical for the initiation of autophagy. Furthermore, ROS play an essential function in the induction of the type III PI3 kinase and autophagy in response to amyloid beta peptide, the main pathogenic mediator of Alzheimer's disease (AD). However, lysosomal blockage also caused by Abeta is independent of ROS. In addition, we demonstrate that autophagy is transcriptionally down-regulated during normal aging in the human brain. Strikingly, in contrast to normal aging, we observe transcriptional up-regulation of autophagy in the brains of AD patients, suggesting that there might be a compensatory regulation of autophagy. Interestingly, we show that an AD drug and an AD drug candidate have inhibitory effects on autophagy, raising the possibility that decreasing input into the lysosomal system may help to reduce cellular stress in AD. Finally, we provide a list of candidate drug targets that can be used to safely modulate levels of autophagy without causing cell death.

Human IRGM Gene "to Be or Not to Be"

Seminars in Immunopathology. Dec, 2010  |  Pubmed ID: 20737271

The immunity-related GTPases (IRG proteins) are one of the strongest early resistance systems against intracellular pathogens. The IRG gene family contains 21 copies arranged as tandem gene clusters on two chromosomes in the C57BL/6 mouse genome but has been reduced to only two copies in humans: IRGC and IRGM. IRGC is not involved in immunity, but the human IRGM gene plays a role in autophagy-targeted destruction of Mycobacterium tuberculosis (BCG) and Salmonella typhimurium. Variant IRGM haplotypes have been associated with increased risk for Crohn's disease and correlated with differential expression of IRGM transcripts. This article reviews in detail the studies performed on human samples, in vitro, and in sequence analyses that provide evidence for the unusual evolutionary history of the IRGM locus and the important role of the IRGM gene in autophagy and Crohn's disease in response to pathogenesis.

LRRK2 is Involved in the IFN-gamma Response and Host Response to Pathogens

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2010  |  Pubmed ID: 20921534

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.

Ubiquitin Accumulation in Autophagy-deficient Mice is Dependent on the Nrf2-mediated Stress Response Pathway: a Potential Role for Protein Aggregation in Autophagic Substrate Selection

The Journal of Cell Biology. Nov, 2010  |  Pubmed ID: 21041446

Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub-Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7(-/-) liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.

Failure and Exploitation of Autophagy in Human Pathologies-cellular Integrity Between Inflammation, Infection, and Cell Survival

Seminars in Immunopathology. Dec, 2010  |  Pubmed ID: 21107575

Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions

PloS One. 2010  |  Pubmed ID: 21217821

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

Proteins Encoded in Genomic Regions Associated with Immune-mediated Disease Physically Interact and Suggest Underlying Biology

PLoS Genetics. 2011  |  Pubmed ID: 21249183

Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in line with observations in Mendelian disease.

Meta-analysis Identifies 29 Additional Ulcerative Colitis Risk Loci, Increasing the Number of Confirmed Associations to 47

Nature Genetics. Mar, 2011  |  Pubmed ID: 21297633

Genome-wide association studies and candidate gene studies in ulcerative colitis have identified 18 susceptibility loci. We conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets, comprising 6,687 cases and 19,718 controls, and followed up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P < 5 × 10(-8)), increasing the number of ulcerative colitis-associated loci to 47. After annotating associated regions using GRAIL, expression quantitative trait loci data and correlations with non-synonymous SNPs, we identified many candidate genes that provide potentially important insights into disease pathogenesis, including IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease risk loci is now 99, including a minimum of 28 shared association signals between Crohn's disease and ulcerative colitis.

Bacterial and Host Determinants of MAL Activation Upon EPEC Infection: the Roles of Tir, ABRA, and FLRT3

PLoS Pathogens. Apr, 2011  |  Pubmed ID: 21490959

Infection of host cells by pathogenic microbes triggers signal transduction pathways leading to a multitude of host cell responses including actin cytoskeletal re-arrangements and transcriptional programs. The diarrheagenic pathogens Enteropathogenic E. coli (EPEC) and the related Enterohemorrhagic E. coli (EHEC) subvert the host-cell actin cytoskeleton to form attaching and effacing lesions on the surface of intestinal epithelial cells by injecting effector proteins via a type III secretion system. Here we use a MAL translocation assay to establish the effect of bacterial pathogens on host cell signaling to transcription factor activation. MAL is a cofactor of Serum response factor (SRF), a transcription factor with important roles in the regulation of the actin cytoskeleton. We show that EPEC induces nuclear accumulation of MAL-GFP. The translocated intimin receptor is essential for this process and phosphorylation of Tyrosine residues 454 and 474 is important. Using an expression screen we identify FLRT3, C22orf28 and TESK1 as novel activators of SRF. Importantly we demonstrate that ABRA (actin-binding Rho-activating protein, also known as STARS) is necessary for EPEC-induced nuclear accumulation of MAL and the novel SRF activator FLRT3, is a component of this pathway. We further demonstrate that ABRA is important for structural maintenance of EPEC pedestals. Our results uncover novel components in pathogen-activated cytoskeleton signalling to MAL activation.

Recurrent Chromosomal Copy Number Alterations in Sporadic Chordomas

PloS One. 2011  |  Pubmed ID: 21602918

The molecular events in chordoma pathogenesis have not been fully delineated, particularly with respect to copy number changes. Understanding copy number alterations in chordoma may reveal critical disease mechanisms that could be exploited for tumor classification and therapy. We report the copy number analysis of 21 sporadic chordomas using array comparative genomic hybridization (CGH). Recurrent copy changes were further evaluated with immunohistochemistry, methylation specific PCR, and quantitative real-time PCR. Similar to previous findings, large copy number losses, involving chromosomes 1p, 3, 4, 9, 10, 13, 14, and 18, were more common than copy number gains. Loss of CDKN2A with or without loss of CDKN2B on 9p21.3 was observed in 16/20 (80%) unique cases of which six (30%) showed homozygous deletions ranging from 76 kilobases to 4.7 megabases. One copy loss of the 10q23.31 region which encodes PTEN was found in 16/20 (80%) cases. Loss of CDKN2A and PTEN expression in the majority of cases was not attributed to promoter methylation. Our sporadic chordoma cases did not show hotspot point mutations in some common cancer gene targets. Moreover, most of these sporadic tumors are not associated with T (brachyury) duplication or amplification. Deficiency of CDKN2A and PTEN expression, although shared across many other different types of tumors, likely represents a key aspect of chordoma pathogenesis. Sporadic chordomas may rely on mechanisms other than copy number gain if they indeed exploit T/brachyury for proliferation.

Leucine-rich Repeat (LRR) Proteins: Integrators of Pattern Recognition and Signaling in Immunity

Autophagy. Sep, 2011  |  Pubmed ID: 21606681

The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2-45 leucine-rich repeats, with each repeat about 20-30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.

MyD88-dependent TLR1/2 Signals Educate Dendritic Cells with Gut-specific Imprinting Properties

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2011  |  Pubmed ID: 21646294

Gut-associated dendritic cells (DC) synthesize all-trans retinoic acid, which is required for inducing gut-tropic lymphocytes. Gut-associated DC from MyD88(-/-) mice, which lack most TLR signals, expressed low levels of retinal dehydrogenases (critical enzymes for all-trans retinoic acid biosynthesis) and were significantly impaired in their ability to induce gut-homing T cells. Pretreatment of extraintestinal DC with a TLR1/2 agonist was sufficient to induce retinal dehydrogenases and to confer these DC with the capacity to induce gut-homing lymphocytes via a mechanism dependent on MyD88 and JNK/MAPK. Moreover, gut-associated DC from TLR2(-/-) mice, or from mice in which JNK was pharmacologically blocked, were impaired in their education to imprint gut-homing T cells, which correlated with a decreased induction of gut-tropic T cells in TLR2(-/-) mice upon immunization. Thus, MyD88-dependent TLR2 signals are necessary and sufficient to educate DC with gut-specific imprinting properties and contribute in vivo to the generation of gut-tropic T cells.

A Francisella Tularensis Locus Required for Spermine Responsiveness is Necessary for Virulence

Infection and Immunity. Sep, 2011  |  Pubmed ID: 21670171

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.

Genetics and Pathogenesis of Inflammatory Bowel Disease

Nature. Jun, 2011  |  Pubmed ID: 21677747

Recent advances have provided substantial insight into the maintenance of mucosal immunity and the pathogenesis of inflammatory bowel disease. Cellular programs responsible for intestinal homeostasis use diverse intracellular and intercellular networks to promote immune tolerance, inflammation or epithelial restitution. Complex interfaces integrate local host and microbial signals to activate appropriate effector programs selectively and even drive plasticity between these programs. In addition, genetic studies and mouse models have emphasized the role of genetic predispositions and how they affect interactions with microbial and environmental factors, leading to pro-colitogenic perturbations of the host-commensal relationship.

HCV Causes Chronic Endoplasmic Reticulum Stress Leading to Adaptation and Interference with the Unfolded Protein Response

PloS One. 2011  |  Pubmed ID: 21949742

The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.

Deep Resequencing of GWAS Loci Identifies Independent Rare Variants Associated with Inflammatory Bowel Disease

Nature Genetics. Nov, 2011  |  Pubmed ID: 21983784

More than 1,000 susceptibility loci have been identified through genome-wide association studies (GWAS) of common variants; however, the specific genes and full allelic spectrum of causal variants underlying these findings have not yet been defined. Here we used pooled next-generation sequencing to study 56 genes from regions associated with Crohn's disease in 350 cases and 350 controls. Through follow-up genotyping of 70 rare and low-frequency protein-altering variants in nine independent case-control series (16,054 Crohn's disease cases, 12,153 ulcerative colitis cases and 17,575 healthy controls), we identified four additional independent risk factors in NOD2, two additional protective variants in IL23R, a highly significant association with a protective splice variant in CARD9 (P < 1 × 10(-16), odds ratio ≈ 0.29) and additional associations with coding variants in IL18RAP, CUL2, C1orf106, PTPN22 and MUC19. We extend the results of successful GWAS by identifying new, rare and probably functional variants that could aid functional experiments and predictive models.

Genome-wide Expression Profiling Implicates a MAST3-regulated Gene Set in Colonic Mucosal Inflammation of Ulcerative Colitis Patients

Inflammatory Bowel Diseases. Oct, 2011  |  Pubmed ID: 21994190

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBDs) presumably caused by dysregulated immune responses to the gut microbiota. Genetic association studies have implicated dozens of chromosomal regions or loci in IBD susceptibility. The next challenge is to explain the individual role of each of these modest effect loci in the disease state. We have previously identified MAST3 as an IBD susceptibility gene through genetic fine-mapping of the 19p linkage region. Testing MAST3 in a reporter assay provided preliminary evidence that MAST3 modulates the activity of inflammation-related transcription factor nuclear factor kappa B. METHODS: Here we characterized the function of MAST3 through an examination of the influence of the modulation of MAST3 expression on endogenous genome-wide expression patterns. More specifically, we looked at differential gene expression resulting from overexpression and knockdown of the MAST3 gene in epithelial and macrophage cell lines. From we highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-jB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. RESULTS: We highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-κB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. These MAST3-regulated genes are central to mucosal immune responses. Among them are proinflammatory cytokines (e.g., CCL20, IL8), regulators of NF-κB (e.g., TNFAIP3, LY96, NFKBIA), genes involved in interferon-induced defense against pathogen invasion (e.g., IFIT1, ISG15), and genes involved in cell adhesion and/or migration (e.g., CD44, TMOD1). CONCLUSIONS: Taken together, these results confirm MAST3 as a modulator of the inflammatory response through regulation of immune gene expression in the gut of IBD patients. (Inflamm Bowel Dis 2011).

Image-based Genome-wide SiRNA Screen Identifies Selective Autophagy Factors

Nature. Dec, 2011  |  Pubmed ID: 22020285

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.

CARMA1 is Necessary for Optimal T Cell Responses in a Murine Model of Allergic Asthma

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2011  |  Pubmed ID: 22075698

CARMA1 is a lymphocyte-specific scaffold protein necessary for T cell activation. Deletion of CARMA1 prevents the development of allergic airway inflammation in a mouse model of asthma due to a defect in naive T cell activation. However, it is unknown if CARMA1 is important for effector and memory T cell responses after the initial establishment of inflammation, findings that would be more relevant to asthma therapies targeted to CARMA1. In the current study, we sought to elucidate the role of CARMA1 in T cells that have been previously activated. Using mice in which floxed CARMA1 exons can be selectively deleted in T cells by OX40-driven Cre recombinase (OX40(+/Cre)CARMA1(F/F)), we report that CD4(+) T cells from these mice have impaired T cell reactivation responses and NF-κB signaling in vitro. Furthermore, in an in vivo recall model of allergic airway inflammation that is dependent on memory T cell function, OX40(+/Cre)CARMA1(F/F) mice have attenuated eosinophilic airway inflammation, T cell activation, and Th2 cytokine production. Using MHC class II tetramers, we demonstrate that the development and maintenance of Ag-specific memory T cells is not affected in OX40(+/Cre)CARMA1(F/F) mice. In addition, adoptive transfer of Th2-polarized OX40(+/Cre)CARMA1(F/F) Ag-specific CD4(+) T cells into wild-type mice induces markedly less airway inflammation in response to Ag challenge than transfer of wild-type Th2 cells. These data demonstrate a novel role for CARMA1 in effector and memory T cell responses and suggest that therapeutic strategies targeting CARMA1 could help treat chronic inflammatory disorders such as asthma.

NLRP3 Inflammasome Plays a Key Role in the Regulation of Intestinal Homeostasis

Inflammatory Bowel Diseases. Jun, 2011  |  Pubmed ID: 20872834

Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18.

Crohn Disease: a Current Perspective on Genetics, Autophagy and Immunity

Autophagy. Apr, 2011  |  Pubmed ID: 20729636

Crohn disease (CD) is a chronic and debilitating inflammatory condition of the gastrointestinal tract. Prevalence in Western populations is 100-150/100,000 and somewhat higher in Ashkenazi Jews. Peak incidence is in early adult life, although any age can be affected and a majority of affected individuals progress to relapsing and chronic disease. Medical treatments rely significantly on empirical corticosteroid therapy and immunosuppression, and intestinal resectional surgery is frequently required. Thus, 80% of patients with CD come to surgery for refractory disease or complications. It is hoped that an improved understanding of pathogenic mechanisms, for example by studying the genetic basis of CD and other forms of inflammatory bowel diseases (IBD), will lead to improved therapies and possibly preventative strategies in individuals identified as being at risk.

Human Leucine-rich Repeat Proteins: a Genome-wide Bioinformatic Categorization and Functional Analysis in Innate Immunity

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2011  |  Pubmed ID: 20616063

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Tribbles 2 (Trib2) is a Novel Regulator of Toll-like Receptor 5 Signaling

Inflammatory Bowel Diseases. Jan, 2012  |  Pubmed ID: 22271508

BACKGROUND: Toll-like receptors (TLRs) are expressed by a variety of cells, including intestinal epithelia. However, the full spectrum of regulators modulating innate responses via TLRs has not been delineated. Tribbles (Trib) have been identified as a highly conserved family of kinase-like proteins. We sought to clarify the role of Trib2 in the TLR signaling pathway. METHODS: Trib2 mRNA and protein levels were analyzed by quantitative polymerase chain reaction (PCR) and western blot, respectively. Immunohistochemical staining was used to determine the expression of Trib2 in human tissue. Involvement of Trib2 in nuclear factor kappa B (NF-κB) pathways was assessed in epithelial cells by NF-κB reporter assay. Proteins that interacted with Trib2 were identified by mass spectrometry and confirmed by immunoprecipitation. The domain essential for Trib2 function was mapped using truncated constructs. RESULTS: Trib2 expression is decreased in active inflamed tissue from patients with inflammatory bowel disease (IBD). Trib2 is expressed in human and mouse colonic epithelium as well as immune cells, and its expression in epithelium is inducible in a ligand-dependent manner by TLR5 ligand stimulation. Trib2 inhibits TLR5-mediated activation of NF-κB downstream of TRAF6. Trib2 selectively modulates mitogen-activated protein kinase (MAPK) pathways p38 and Jun N-terminal kinase (JNK) but not p44/p42 (ERK1/2). NF-κB2 (p100) was identified as a Trib2 binding partner in regulating the TLR5 signaling pathway that leads to inhibition of NF-κB activity. Residues 158-177 in the Trib2 kinase-like domain are required for Trib2 function. CONCLUSIONS: These observations indicate that Trib2 is a novel regulator in the TLR5 signaling pathway and altered expression of Trib2 may play a role in IBD. (Inflamm Bowel Dis 2012;).

Genome-wide SiRNA Screen for Mediators of NF-κB Activation

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2012  |  Pubmed ID: 22308454

Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β-, or TNFα-mediated canonical NFκB activation.