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1.
EMBO J ; 38(20): e101266, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31544965

RESUMEN

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1ß by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1ß generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1ß-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.


Asunto(s)
Caspasa 1/metabolismo , Vesículas Extracelulares/metabolismo , Inflamasomas/inmunología , Macrófagos/inmunología , Peroxirredoxinas/fisiología , Choque Séptico/prevención & control , Animales , Caspasa 1/genética , Citocinas/metabolismo , Femenino , Inflamasomas/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Choque Séptico/inducido químicamente , Choque Séptico/inmunología , Choque Séptico/patología , Transducción de Señal
2.
Gastroenterology ; 159(4): 1357-1374.e10, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32673694

RESUMEN

BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.


Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Células Epiteliales/patología , Íleon/metabolismo , Íleon/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Animales , Autofagia , Células CACO-2 , Técnicas de Cultivo de Célula , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Humanos , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Ratones , Transducción de Señal
3.
Bioinformatics ; 29(13): 1600-6, 2013 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23620359

RESUMEN

MOTIVATION: Protocols to generate strand-specific transcriptomes with next-generation sequencing platforms have been used by the scientific community roughly since 2008. Strand-specific reads allow for detection of antisense events and a higher resolution of expression profiles enabling extension of current transcript annotations. However, applications making use of this strandedness information are still scarce. RESULTS: Here we present a tool (Janus), which focuses on the identification of transcriptional active regions in antisense orientation to known and novel transcribed elements of the genome. Janus can compare the antisense events of multiple samples and assigns scores to identify mutual expression of either transcript in a sense/antisense pair, which could hint to regulatory mechanisms. Janus is able to make use of single-nucleotide variant (SNV) and methylation data, if available, and reports the sense to antisense ratio of regions in the vicinity of the identified genetic and epigenetic variation. Janus interrogates positions of heterozygous SNVs to identify strand-specific allelic imbalance. AVAILABILITY: Janus is written in C/C++ and freely available at http://www.ikmb.uni-kiel.de/janus/janus.html under terms of GNU General Public License, for both, Linux and Windows 64×. Although the binaries will work without additional downloads, the software depends on bamtools (https://github.com/pezmaster31/bamtools) for compilation. A detailed tutorial section is included in the first section of the supplemental material and included as brief readme.txt in the tutorial archive. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , ARN sin Sentido/biosíntesis , Programas Informáticos , Línea Celular Transformada , Metilación de ADN , Variación Genética , Humanos
4.
Nat Commun ; 13(1): 6266, 2022 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-36271073

RESUMEN

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.


Asunto(s)
ADN Metiltransferasa 3A , Enfermedades Inflamatorias del Intestino , Humanos , Ratones , Animales , Células CACO-2 , Mucosa Intestinal/metabolismo , Colon/patología , Células Epiteliales/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Factores de Necrosis Tumoral/metabolismo , ADN/metabolismo
5.
Oncotarget ; 9(16): 12941-12958, 2018 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-29560122

RESUMEN

The disintegrin metalloprotease ADAM17 has a critical role in intestinal inflammation and regeneration in mice, as illustrated by the dramatically increased susceptibility of ADAM17 hypomorphic (ADAM17ex/ex) mice to dextran sulfate sodium (DSS)-induced colitis. Similarly, necroptosis has been implicated in inflammatory responses in the intestine. In this study, we have investigated the contribution of necroptosis to ADAM17-regulated intestinal inflammation in vivo by crossing ADAM17ex/ex mice with mice that lack the necroptotic core protein RIPK3. Despite the loss of RIPK3, ADAM17ex/ex/RIPK3-/- mice showed the same increased susceptibility as ADAM17ex/ex mice in both acute and chronic models of DSS-induced colitis. Mice of both genotypes revealed comparable results with regard to weight loss, disease activity index and colitis-associated changes of inner organs. Histopathological analyses confirmed similar tissue destruction, loss of barrier integrity, immune cell infiltration, and cell death; serum analyses revealed similar levels of the pro-inflammatory cytokine KC. Resolving these unexpected findings, ADAM17ex/ex mice did not show phosphorylation of RIPK3 and its necroptotic interaction partner MLKL during DSS-induced colitis, although both proteins were clearly expressed. Consistent with these findings, murine embryonic fibroblasts derived from ADAM17ex/ex mice were protected from tumor necrosis factor (TNF)-induced necroptosis and failed to show phosphorylation of MLKL and RIPK3 after induction of necroptosis by TNF, revealing a novel, undescribed role of the protease ADAM17 in necroptosis.

6.
Genome Med ; 10(1): 27, 2018 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-29653584

RESUMEN

BACKGROUND: The interplay of epigenetic processes and the intestinal microbiota may play an important role in intestinal development and homeostasis. Previous studies have established that the microbiota regulates a large proportion of the intestinal epithelial transcriptome in the adult host, but microbial effects on DNA methylation and gene expression during early postnatal development are still poorly understood. Here, we sought to investigate the microbial effects on DNA methylation and the transcriptome of intestinal epithelial cells (IECs) during postnatal development. METHODS: We collected IECs from the small intestine of each of five 1-, 4- and 12 to 16-week-old mice representing the infant, juvenile, and adult states, raised either in the presence or absence of a microbiota. The DNA methylation profile was determined using reduced representation bisulfite sequencing (RRBS) and the epithelial transcriptome by RNA sequencing using paired samples from each individual mouse to analyze the link between microbiota, gene expression, and DNA methylation. RESULTS: We found that microbiota-dependent and -independent processes act together to shape the postnatal development of the transcriptome and DNA methylation signatures of IECs. The bacterial effect on the transcriptome increased over time, whereas most microbiota-dependent DNA methylation differences were detected already early after birth. Microbiota-responsive transcripts could be attributed to stage-specific cellular programs during postnatal development and regulated gene sets involved primarily immune pathways and metabolic processes. Integrated analysis of the methylome and transcriptome data identified 126 genomic loci at which coupled differential DNA methylation and RNA transcription were associated with the presence of intestinal microbiota. We validated a subset of differentially expressed and methylated genes in an independent mouse cohort, indicating the existence of microbiota-dependent "functional" methylation sites which may impact on long-term gene expression signatures in IECs. CONCLUSIONS: Our study represents the first genome-wide analysis of microbiota-mediated effects on maturation of DNA methylation signatures and the transcriptional program of IECs after birth. It indicates that the gut microbiota dynamically modulates large portions of the epithelial transcriptome during postnatal development, but targets only a subset of microbially responsive genes through their DNA methylation status.


Asunto(s)
Metilación de ADN/genética , Células Epiteliales/metabolismo , Microbioma Gastrointestinal/genética , Regulación del Desarrollo de la Expresión Génica , Intestinos/citología , Transcriptoma/genética , Animales , Femenino , Sitios Genéticos , Crecimiento y Desarrollo/genética , Ratones Endogámicos C57BL , ARN/genética , Transcripción Genética
7.
J Exp Med ; 215(11): 2868-2886, 2018 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-30254094

RESUMEN

A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1 ΔIEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1 ΔIEC and Atg16l1 ΔIEC/Xbp1 ΔIEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22-induced ileal inflammation in Atg16l1 ΔIEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium.


Asunto(s)
Proteínas Relacionadas con la Autofagia/inmunología , Proteínas Portadoras/inmunología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Proteínas de la Membrana/inmunología , Nucleotidiltransferasas/inmunología , Transducción de Señal/inmunología , Animales , Proteínas Relacionadas con la Autofagia/genética , Células CACO-2 , Proteínas Portadoras/genética , Variación Genética , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Interleucinas/genética , Mucosa Intestinal/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Nucleotidiltransferasas/genética , Transducción de Señal/genética , Interleucina-22
9.
Nat Commun ; 8(1): 2063, 2017 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-29234056

RESUMEN

FOXO3 is consistently annotated as a human longevity gene. However, functional variants and underlying mechanisms for the association remain unknown. Here, we perform resequencing of the FOXO3 locus and single-nucleotide variant (SNV) genotyping in three European populations. We find two FOXO3 SNVs, rs12206094 and rs4946935, to be most significantly associated with longevity and further characterize them functionally. We experimentally validate the in silico predicted allele-dependent binding of transcription factors (CTCF, SRF) to the SNVs. Specifically, in luciferase reporter assays, the longevity alleles of both variants show considerable enhancer activities that are reversed by IGF-1 treatment. An eQTL database search reveals that the alleles are also associated with higher FOXO3 mRNA expression in various human tissues, which is in line with observations in long-lived model organisms. In summary, we present experimental evidence for a functional link between common intronic variants in FOXO3 and human longevity.


Asunto(s)
Proteína Forkhead Box O3/fisiología , Longevidad/genética , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genética , Factores de Edad , Anciano , Anciano de 80 o más Años , Alelos , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Simulación por Computador , Femenino , Proteína Forkhead Box O3/genética , Haplotipos/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Intrones/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo
10.
Cell Rep ; 16(8): 2208-2218, 2016 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-27524624

RESUMEN

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.


Asunto(s)
Colitis/inmunología , Disbiosis/inmunología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Receptores de Interleucina/inmunología , Animales , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/microbiología , Sulfato de Dextran , Disbiosis/tratamiento farmacológico , Disbiosis/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Regulación de la Expresión Génica , Granulocitos/efectos de los fármacos , Granulocitos/inmunología , Granulocitos/microbiología , Interleucina-23/farmacología , Interleucinas/genética , Interleucinas/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Isoanticuerpos/farmacología , Masculino , Ratones , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Proteínas Asociadas a Pancreatitis/genética , Proteínas Asociadas a Pancreatitis/inmunología , Proteínas Asociadas a Pancreatitis/farmacología , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Transducción de Señal , Células Madre/efectos de los fármacos , Células Madre/inmunología , Células Madre/microbiología , Interleucina-22
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