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1.
Gut Microbes ; 16(1): 2350150, 2024.
Article En | MEDLINE | ID: mdl-38841888

Comensal Bacteroidota (Bacteroidota) and Enterobacteriacea are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism in Bacteroidota remain unclear. By using the classical lipopolysaccharide/O-antigen 'rfb operon' in Enterobacteriaceae as a surface antigen model (5-rfb-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification, we characterized the integrity and conservancy of the entire rfb operon in Bacteroidota. Through exploratory analysis of complete genomes and metagenomes, we discovered that most Bacteroidota have the rfb operon fragmented into nonrandom patterns of gene-singlets and doublets/triplets, termed 'rfb-gene-clusters', or rfb-'minioperons' if predicted as transcriptional. To reflect global operon integrity, contiguity, duplication, and fragmentation principles, we propose a six-category (infra/supra-numerary) cataloging system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in gut-wall specific micro-niches or micropathologies. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes. DNA insertions, overrepresenting DNA-exchange-avid (Bacteroides) species, impact our interpretation of functional metagenomics data by inflating by inflating gene-based pathway inference and by overestimating 'extra-species' abundance. Of disease relevance, Bacteroidota species isolated from cavitating/cavernous fistulous tract (CavFT) microlesions in Crohn's Disease have supra-numerary fragmented operons, stimulate TNF-alpha from macrophages with low potency, and do not induce hyperacute peritonitis in mice compared to CavFT Enterobacteriaceae. The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism/opportunism requires further studies to elucidate their potential for novel diagnostics and therapeutics, and to elucidate the role of co-existing pathobionts in Crohn's disease microlesions.


Crohn Disease , Gastrointestinal Microbiome , Metagenomics , Operon , Mice , Animals , Humans , Crohn Disease/microbiology , Crohn Disease/genetics , Bacteroidetes/genetics , Bacteroidetes/classification , Antigens, Bacterial/genetics , Genome, Bacterial , Enterobacteriaceae/genetics , Enterobacteriaceae/classification
2.
Sci Transl Med ; 16(732): eadg7895, 2024 Jan 31.
Article En | MEDLINE | ID: mdl-38295187

A mutation in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Patients with ALS or FTD often develop autoimmunity and inflammation that precedes or coincides with the onset of neurological symptoms, but the underlying mechanisms are poorly understood. Here, we knocked out murine C9orf72 in seven hematopoietic progenitor compartments by conditional mutagenesis and found that myeloid lineage C9orf72 prevents splenomegaly, loss of tolerance, and premature mortality. Furthermore, we demonstrated that C9orf72 plays a role in lymphoid cells to prevent interleukin-17A (IL-17A) production and neutrophilia. Mass cytometry identified early and sustained elevation of the costimulatory molecule CD80 expressed on C9orf72-deficient mouse macrophages, monocytes, and microglia. Enrichment of CD80 was similarly observed in human spinal cord microglia from patients with C9ORF72-mediated ALS compared with non-ALS controls. Single-cell RNA sequencing of murine spinal cord, brain cortex, and spleen demonstrated coordinated induction of gene modules related to antigen processing and presentation and antiviral immunity in C9orf72-deficient endothelial cells, microglia, and macrophages. Mechanistically, C9ORF72 repressed the trafficking of CD80 to the cell surface in response to Toll-like receptor agonists, interferon-γ, and IL-17A. Deletion of Il17a in C9orf72-deficient mice prevented CD80 enrichment in the spinal cord, reduced neutrophilia, and reduced gut T helper type 17 cells. Last, systemic delivery of an IL-17A neutralizing antibody augmented motor performance and suppressed neuroinflammation in C9orf72-deficient mice. Altogether, we show that C9orf72 orchestrates myeloid costimulatory potency and provide support for IL-17A as a therapeutic target for neuroinflammation associated with ALS or FTD.


Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Animals , Humans , Mice , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , C9orf72 Protein/genetics , Interleukin-17 , Neuroinflammatory Diseases , Endothelial Cells/metabolism
3.
Bioinformatics ; 39(8)2023 08 01.
Article En | MEDLINE | ID: mdl-37527009

SUMMARY: Microbiome research is now moving beyond the compositional analysis of microbial taxa in a sample. Increasing evidence from large human microbiome studies suggests that functional consequences of changes in the intestinal microbiome may provide more power for studying their impact on inflammation and immune responses. Although 16S rRNA analysis is one of the most popular and a cost-effective method to profile the microbial compositions, marker-gene sequencing cannot provide direct information about the functional genes that are present in the genomes of community members. Bioinformatic tools have been developed to predict microbiome function with 16S rRNA gene data. Among them, PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) has become one of the most popular functional profile prediction tools, which generates community-wide pathway abundances. However, no state-of-art inference tools are available to test the differences in pathway abundances between comparison groups. We have developed ggpicrust2, an R package, for analyzing functional profiles derived from 16S rRNA sequencing. This powerful tool enables researchers to conduct extensive differential abundance analyses and generate visually appealing visualizations that effectively highlight functional signals. With ggpicrust2, users can obtain publishable results and gain deeper insights into the functional composition of their microbial communities. AVAILABILITY AND IMPLEMENTATION: The package is open-source under the MIT and file license and is available at CRAN and https://github.com/cafferychen777/ggpicrust2. Its shiny web is available at https://a95dps-caffery-chen.shinyapps.io/ggpicrust2_shiny/.


Gastrointestinal Microbiome , Microbiota , Humans , Software , RNA, Ribosomal, 16S/genetics , Phylogeny
4.
bioRxiv ; 2023 Jun 02.
Article En | MEDLINE | ID: mdl-37398285

The causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism within the phylum Bacteroidota remain unclear (1, 2). Using the classical lipopolysaccharide/O-antigen 'rfb operon' in Enterobacteriaceae as a surface antigen model (5-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification (3), we characterized the architecture/conservancy of the entire rfb operon in Bacteroidota. Analyzing complete genomes, we discovered that most Bacteroidota have the rfb operon fragmented into non-random gene-singlets and/or doublets/triplets, termed 'minioperons'. To reflect global operon integrity, duplication, and fragmentation principles, we propose a five-category (infra/supernumerary) cataloguing system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in specific micro-niches. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes (4). DNA insertions overrepresenting DNA-exchange-avid species, impact functional metagenomics by inflating gene-based pathway inference and overestimating 'extra-species' abundance. Using bacteria from inflammatory gut-wall cavernous micro-tracts (CavFT) in Crohn's Disease (5), we illustrate that bacteria with supernumerary-fragmented operons cannot produce O-antigen, and that commensal/CavFT Bacteroidota stimulate macrophages with lower potency than Enterobacteriaceae, and do not induce peritonitis in mice. The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism offers potential for novel diagnostics and therapeutics.

6.
Neuron ; 110(10): 1671-1688.e6, 2022 05 18.
Article En | MEDLINE | ID: mdl-35294901

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration accompanied by aberrant accumulation and loss of function of the RNA-binding protein TDP43. Thus far, it remains unresolved to what extent TDP43 loss of function directly contributes to motor system dysfunction. Here, we employed gene editing to find whether the mouse ortholog of the TDP43-regulated gene STMN2 has an important function in maintaining the motor system. Both mosaic founders and homozygous loss-of-function Stmn2 mice exhibited neuromuscular junction denervation and fragmentation, resulting in muscle atrophy and impaired motor behavior, accompanied by an imbalance in neuronal microtubule dynamics in the spinal cord. The introduction of human STMN2 through BAC transgenesis was sufficient to rescue the motor phenotypes observed in Stmn2 mutant mice. Collectively, our results demonstrate that disrupting the ortholog of a single TDP43-regulated RNA is sufficient to cause substantial motor dysfunction, indicating that disruption of TDP43 function is likely a contributor to ALS.


Amyotrophic Lateral Sclerosis , Stathmin , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Disease Models, Animal , Homozygote , Mice , Mice, Transgenic , Motor Neurons/metabolism , Neuromuscular Junction/metabolism , Stathmin/genetics , Stathmin/metabolism
7.
Nature ; 582(7810): 89-94, 2020 06.
Article En | MEDLINE | ID: mdl-32483373

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.


C9orf72 Protein/genetics , Gastrointestinal Microbiome/physiology , Gliosis/microbiology , Gliosis/pathology , Inflammation/genetics , Inflammation/microbiology , Spinal Cord/pathology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Anti-Bacterial Agents/pharmacology , Autoimmunity/drug effects , Autoimmunity/genetics , Autoimmunity/immunology , Cell Movement/drug effects , Cytokines/immunology , Fecal Microbiota Transplantation , Female , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/immunology , Gliosis/genetics , Gliosis/prevention & control , Inflammation/pathology , Inflammation/prevention & control , Loss of Function Mutation/genetics , Male , Mice , Microglia/immunology , Microglia/microbiology , Microglia/pathology , Spinal Cord/immunology , Spinal Cord/microbiology , Survival Rate
8.
Nat Neurosci ; 22(2): 167-179, 2019 02.
Article En | MEDLINE | ID: mdl-30643292

The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.


Amyotrophic Lateral Sclerosis/metabolism , DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , Motor Neurons/metabolism , Axons/metabolism , Cell Line , Down-Regulation , Female , Humans , Induced Pluripotent Stem Cells , Male , Spinal Cord/metabolism , Stathmin
9.
Genes Dev ; 32(13-14): 929-943, 2018 07 01.
Article En | MEDLINE | ID: mdl-29950492

While a mutation in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated Smcr8 loss-of-function mutant mice and found that they developed phenotypes also observed in C9orf72 loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in Smcr8 mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components-phenotypes that we subsequently observed in C9orf72 loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis-processes of potential importance to ALS.


Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Autoimmunity/genetics , Carrier Proteins/metabolism , Exocytosis/genetics , Lysosomes/metabolism , Animals , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Carrier Proteins/genetics , Gene Expression Regulation/genetics , Humans , Lymph Nodes/pathology , Lysosomal-Associated Membrane Protein 1/genetics , Macrophages/pathology , Mice , Mice, Knockout , Mutation , Protein Isoforms , Protein Stability , Splenomegaly/genetics
10.
Sci Transl Med ; 8(347): 347ra93, 2016 07 13.
Article En | MEDLINE | ID: mdl-27412785

C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.


Autoimmune Diseases/etiology , Autoimmune Diseases/genetics , C9orf72 Protein/genetics , Animals , Autoimmune Diseases/metabolism , Autoimmunity/genetics , Autoimmunity/physiology , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/physiology , Cytokines/metabolism , Leukemia/genetics , Leukemia/metabolism , Mice , Mutation/genetics , Splenomegaly/genetics , Splenomegaly/immunology , Thrombocytopenia/genetics , Thrombocytopenia/immunology
11.
J Biol Chem ; 289(41): 28213-24, 2014 Oct 10.
Article En | MEDLINE | ID: mdl-25170077

NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8.


Crohn Disease/genetics , Endopeptidases/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Interleukin-8/genetics , Macrophages/metabolism , Nod2 Signaling Adaptor Protein/genetics , RNA, Small Interfering/genetics , Ubiquitin Thiolesterase/genetics , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Line, Transformed , Crohn Disease/metabolism , Crohn Disease/pathology , Endopeptidases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Genetic Loci , Genome-Wide Association Study , HEK293 Cells , High-Throughput Screening Assays , Humans , Interleukin-8/agonists , Interleukin-8/antagonists & inhibitors , Interleukin-8/metabolism , Macrophages/pathology , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/antagonists & inhibitors , Nod2 Signaling Adaptor Protein/metabolism , Protein Interaction Mapping , Protein Transport , RNA Stability , RNA, Small Interfering/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitination
12.
Cell Host Microbe ; 15(6): 779-91, 2014 Jun 11.
Article En | MEDLINE | ID: mdl-24882704

Adult hematopoietic stem cells (HSCs) are maintained in specialized niches within the bone marrow under steady-state conditions and mobilize for extramedullary hematopoiesis during periods of stress such as bacterial infections. However, the underlying mechanisms are unclear. We show that systemic infection of mice with Escherichia coli, commonly associated with bacteremia in humans, mobilizes functional HSCs to the spleen. Accumulation of splenic HSCs (CD150+CD48-Lin(-/low)Sca1+cKit+) was diminished in TLR4-deficient and RIPK2-deficient mice, implicating TLRs and cytosolic NOD1/NOD2 signaling in the process. Accordingly, dual stimulation of NOD1 and TLR4 in radio-resistant cells alone was sufficient to mobilize HSCs, while TLR4 expression on HSCs was dispensable. Mechanistically, TLR4 and NOD1 synergistically induced granulocyte colony-stimulating factor (G-CSF), which was required for extramedullary HSC accumulation. Mobilized HSCs and progenitor cells gave rise to neutrophils and monocytes and contributed to limiting secondary infection.


Escherichia coli Infections/metabolism , Hematopoietic Stem Cells/microbiology , Nod1 Signaling Adaptor Protein/metabolism , Toll-Like Receptor 4/metabolism , Animals , Antigens, CD/metabolism , Ataxin-10 , Bone Marrow/microbiology , Bone Marrow/pathology , CD48 Antigen , Carrier Proteins/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Infections/pathology , Escherichia coli K12/pathogenicity , Granulocyte Colony-Stimulating Factor/metabolism , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells/metabolism , Mice, Inbred C57BL , Mice, Mutant Strains , Nod1 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/metabolism , Receptors, Cell Surface/metabolism , Signaling Lymphocytic Activation Molecule Family Member 1 , Spleen/microbiology , Spleen/pathology , Toll-Like Receptor 4/genetics
13.
J Biol Chem ; 289(2): 1142-50, 2014 Jan 10.
Article En | MEDLINE | ID: mdl-24265316

The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-methylenedioxy-ß-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 (absent in melanoma 2) inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speck formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry, and mutation studies suggest that both the nucleotide binding oligomerization domain and the leucine-rich repeat domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.


Carrier Proteins/metabolism , Dioxolanes/pharmacology , Inflammasomes/metabolism , Macrophages/drug effects , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Blotting, Western , Carrier Proteins/genetics , Cells, Cultured , Dioxolanes/chemistry , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Inflammasomes/genetics , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence , Molecular Structure , NLR Family, Pyrin Domain-Containing 3 Protein , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Syk Kinase , Tumor Necrosis Factor-alpha/metabolism
14.
Sci Signal ; 6(258): rs3, 2013 Jan 15.
Article En | MEDLINE | ID: mdl-23322906

The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor κB (NF-κB) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn's disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn's disease risk were identified in the screen. A comparison of candidates from this screen with other "omics" data sets revealed interconnected networks of genes implicated in NF-κB signaling, thus supporting a role for NOD2 and NF-κB pathways in the pathogenesis of Crohn's disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-κB. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor-α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB-mediated inflammation.


Genome, Human/genetics , NF-kappa B/genetics , Nod2 Signaling Adaptor Protein/genetics , RNA, Small Interfering/genetics , Signal Transduction/genetics , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Animals , Blotting, Western , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Survival/genetics , Cells, Cultured , Crohn Disease/genetics , Gene Expression Regulation/drug effects , Gene Regulatory Networks/drug effects , HEK293 Cells , Humans , I-kappa B Proteins/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Models, Genetic , NADPH Oxidases/deficiency , NADPH Oxidases/genetics , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/metabolism , RNA Interference , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology
15.
Arthritis Res Ther ; 15(6): R186, 2013 Nov 12.
Article En | MEDLINE | ID: mdl-24456966

INTRODUCTION: Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model. METHODS: DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression. RESULTS: Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1ß, IL-6, and IL-22 in the CFA/CII group and IL-1ß, tumor necrosis factor-α, transforming growth factor-ß, IL-6 and IL-23 in the IFA/CII group. CONCLUSIONS: Chronic P. gingivalis oral infection prior to arthritis induction increases the immune system activation favoring Th17 cell responses, and ultimately accelerating arthritis development. These results suggest that chronic oral infection may influence RA development mainly through activation of Th17-related pathways.


Arthritis, Experimental/microbiology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/microbiology , Arthritis, Rheumatoid/pathology , Bacteroidaceae Infections/complications , Animals , Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , Cytokines/analysis , Cytokines/blood , Male , Mice , Mice, Inbred DBA , Porphyromonas gingivalis
16.
Nat Immunol ; 13(5): 449-56, 2012 May.
Article En | MEDLINE | ID: mdl-22484733

Intestinal phagocytes transport oral antigens and promote immune tolerance, but their role in innate immune responses remains unclear. Here we found that intestinal phagocytes were anergic to ligands for Toll-like receptors (TLRs) or commensals but constitutively expressed the precursor to interleukin 1ß (pro-IL-1ß). After infection with pathogenic Salmonella or Pseudomonas, intestinal phagocytes produced mature IL-1ß through the NLRC4 inflammasome but did not produce tumor necrosis factor (TNF) or IL-6. BALB/c mice deficient in NLRC4 or the IL-1 receptor were highly susceptible to orogastric but not intraperitoneal infection with Salmonella. That enhanced lethality was preceded by impaired expression of endothelial adhesion molecules, lower neutrophil recruitment and poor intestinal pathogen clearance. Thus, NLRC4-dependent production of IL-1ß by intestinal phagocytes represents a specific response that discriminates pathogenic bacteria from commensal bacteria and contributes to host defense in the intestine.


Apoptosis Regulatory Proteins/immunology , Calcium-Binding Proteins/immunology , Clonal Anergy , Host-Pathogen Interactions/immunology , Interleukin-1beta/metabolism , Intestines/immunology , Intestines/microbiology , Phagocytes/immunology , Animals , Apoptosis Regulatory Proteins/genetics , Calcium-Binding Proteins/genetics , Caspase 1/metabolism , Flagellin/immunology , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-6/biosynthesis , Interleukin-6/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Monocytes/metabolism , Neutrophil Infiltration/genetics , Neutrophil Infiltration/immunology , Neutrophils/immunology , Neutrophils/pathology , Phagocytes/microbiology , Pseudomonas/immunology , Pseudomonas Infections/immunology , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Salmonella/genetics , Salmonella/immunology , Salmonella Infections/genetics , Salmonella Infections/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology
17.
Gastroenterology ; 141(3): 1003-1013.e1-10, 2011 Sep.
Article En | MEDLINE | ID: mdl-21699772

BACKGROUND & AIMS: Adenomatous polyps are precursors to colorectal cancer (CRC), whereas hyperplastic polyps (HPPs) have low risk of progression to CRC. Mutations in KRAS are found in ∼40% of CRCs and large adenomas and a subset of HPPs. We investigated the reasons why HPPs with KRAS mutations lack malignant potential and compared the effects of Kras/KRAS activation with those of Apc/APC inactivation, which promotes adenoma formation. METHODS: We activated a KrasG12D mutant allele or inactivated Apc alleles in mouse colon epithelium and analyzed phenotypes and expression of selected genes and proteins. The mouse data were validated using samples of human HPPs and adenomas. Signaling pathways and factors contributing to Kras/KRAS-induced phenotypes were studied in intestinal epithelial cells. RESULTS: Activation of Kras led to hyperplasia and serrated crypt architecture akin to that observed in human HPPs. We also observed loss of Paneth cells and increases in goblet cell numbers. Abnormalities in Kras-mediated differentiation and proliferation required mitogen-activated protein kinase signaling and were linked to activation of the Hes1 transcription factor. Human HPPs also had activation of HES1. In contrast to Apc/APC inactivation, Kras/KRAS activation did not increase expression of crypt stem cell markers in colon epithelium or colony formation in vitro. Kras/KRAS activation was not associated with substantial induction of p16(INK4a) protein expression in mouse colon epithelium or human HPPs. CONCLUSIONS: Although Kras/KRAS mutation promotes serrated and hyperplastic morphologic features in colon epithelium, it is not able to initiate adenoma development, perhaps in part because activated Kras/KRAS signaling does not increase the number of presumptive stem cells in affected crypts.


Cell Differentiation/physiology , Colon/pathology , Intestinal Mucosa/pathology , Mutation/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/physiology , Stem Cells/pathology , Adenoma/pathology , Adenoma/physiopathology , Animals , Basic Helix-Loop-Helix Transcription Factors/physiology , Cell Line , Cell Proliferation , Colon/physiology , Colonic Polyps/pathology , Colonic Polyps/physiopathology , Colorectal Neoplasms/pathology , Colorectal Neoplasms/physiopathology , Disease Models, Animal , Disease Progression , Homeodomain Proteins/physiology , Humans , Hyperplasia , Intestinal Mucosa/physiology , Mice , Mice, Transgenic , Signal Transduction/physiology , Transcription Factor HES-1
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