RESUMEN
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.
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Enfermedad de Crohn , Microbioma Gastrointestinal , Metagenómica , Operón , Ratones , Animales , Humanos , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/genética , Bacteroidetes/genética , Bacteroidetes/clasificación , Antígenos Bacterianos/genética , Genoma Bacteriano , Enterobacteriaceae/genética , Enterobacteriaceae/clasificaciónRESUMEN
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.
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Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Demencia Frontotemporal , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Células Endoteliales/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Interleucina-17 , Enfermedades NeuroinflamatoriasRESUMEN
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.
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Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie NEC is lacking. This study aimed at filling in this gap. We combine single-cell RNA sequencing (scRNAseq), T-cell receptor beta (TCRß) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions, and zonal changes in NEC. We find an abundance of proinflammatory macrophages, fibroblasts, endothelial cells as well as T cells that exhibit increased TCRß clonal expansion. Villus tip epithelial cells are reduced in NEC and the remaining epithelial cells up-regulate proinflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that are associated with inflammation in NEC mucosa. Our analyses highlight the cellular dysregulations of NEC-associated intestinal tissue and identify potential targets for biomarker discovery and therapeutics.
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Enterocolitis Necrotizante , Lactante , Recién Nacido , Humanos , Enterocolitis Necrotizante/genética , Células Endoteliales , Intestino Delgado , Recien Nacido Prematuro , Intestinos , Mucosa IntestinalRESUMEN
Glycogen Storage Disease type 1b (GSD1b) is a rare disease manifesting as hypoglycemia, recurrent infections and neutropenia, resulting from deleterious mutations in the SLC37A4 gene encoding the glucose-6-phosphate transporter. The susceptibility to infections is thought to be attributed not only to the neutrophil defect, though extensive immunophenotyping characterization is currently missing. Here we apply a systems immunology approach utilizing Cytometry by Time Of Flight (CyTOF) to map the peripheral immune landscape of 6 GSD1b patients. When compared to control subjects, those with GSD1b had a significant reduction in anti-inflammatory macrophages, CD16+ macrophages, and Natural Killer cells. Additionally, there was a preference towards a central versus an effector memory phenotype in multiple T cell populations, which may suggest that these changes stem from an inability of activated immune cell populations to undergo the appropriate switch to glycolytic metabolism in the hypoglycemic conditions associated with GSD1b. Furthermore, we identified a global reduction of CD123, CD14, CCR4, CD24 and CD11b across several populations and a multi-cluster upregulation of CXCR3, hinting at a potential role of impaired immune cell trafficking in the context of GSD1b. Taken together, our data indicates that that the immune impairment observed in GSD1b patients extends far beyond neutropenia and encompasses innate and adaptive compartments, which may provide novel insights into the pathogenesis of this disorder.
RESUMEN
Purpose: Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulator of necroptosis and inflammatory responses. We present the clinical features, genetic analysis and immune work-up of two patients with infantile-onset inflammatory bowel disease (IBD) resulting from RIPK1 mutations. Methods: Whole exome and Sanger sequencing was performed in two IBD patients. Mass cytometry time of flight (CyTOF) was conducted for in-depth immunophenotyping on one of the patient's peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn's disease. Results: The patients presented with severe colitis and perianal fistulas in the first months of life, without severe/atypical infections. Genetic studies identified pathogenic genetic variants in RIPK1 (Patient 1, A c.1934C>T missense mutation in Exon 11; Patient 2, c.580G>A missense mutation residing in Exon 4). Protein modeling demonstrated that the mutation in Patient 1 displaces a water molecule, potentially disrupting the local environment, and the mutation in Patient 2 may lead to disruption of the packing and conformation of the kinase domain. Immunofluorescence RIPK1 staining in rectal biopsies demonstrated no expression for Patient 1 and minimal expression for Patient 2, compared to controls and patients with active Crohn's disease. Using CyTOF unbiased clustering analysis, we identified peripheral immune dysregulation in one of these patients, characterized by an increase in IFNγ CD8+ T cells along with a decrease in monocytes, dendritic cells and B cells. Moreover, RIPK1-deficient patient's immune cells exhibited decreased IL-6 production in response to lipopolysaccharide (LPS) across multiple cell types including T cells, B cells and innate immune cells. Conclusions: Mutations in RIPK1 should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1's role in inflammasome activation, but also in epithelial cells, it is unclear whether IL1 blockade or allogeneic hematopoietic stem cell transplantation can suppress or cure the hyper-inflammatory response in these patients. Additional studies in humans are required to better define the role of RIPK1 in regulating intestinal immune responses, and how treatment can be optimized for patients with RIPK1 deficiency.
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Colitis , Enfermedad de Crohn , Fístula , Enfermedades Inflamatorias del Intestino , Humanos , Enfermedad de Crohn/genética , Leucocitos Mononucleares , Linfocitos T CD8-positivos , Enfermedades Inflamatorias del Intestino/genética , Mutación , Enfermedad Crónica , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Defective DNA repair pathways contribute to the development of chronic kidney disease (CKD) in humans. However, the molecular mechanisms underlying DNA damage-induced CKD pathogenesis are not well understood. Here, we investigated the role of tubular cell DNA damage in the pathogenesis of CKD using mice in which the DNA repair protein Fan1 was knocked out. The phenotype of these mice is orthologous to the human DNA damage syndrome, karyomegalic interstitial nephritis (KIN). Inactivation of Fan1 in kidney proximal tubule cells sensitized the kidneys to genotoxic and obstructive injury characterized by replication stress and persistent DNA damage response activity. Accumulation of DNA damage in Fan1 tubular cells induced epithelial dedifferentiation and tubular injury. Characteristic to KIN, cells with chronic DNA damage failed to complete mitosis and underwent polyploidization. In vitro and in vivo studies showed that polyploidization was caused by the overexpression of DNA replication factors CDT1 and CDC6 in FAN1 deficient cells. Mechanistically, inhibiting DNA replication with Roscovitine reduced tubular injury, blocked the development of KIN and mitigated kidney function in these Fan1 knockout mice. Thus, our data delineate a mechanistic pathway by which persistent DNA damage in the kidney tubular cells leads to kidney injury and development of CKD. Furthermore, therapeutic modulation of cell cycle activity may provide an opportunity to mitigate the DNA damage response induced CKD progression.
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Nefritis Intersticial , Insuficiencia Renal Crónica , Animales , Humanos , Ratones , Daño del ADN , Reparación del ADN , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Fibrosis , Riñón/patología , Ratones Noqueados , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Nefritis Intersticial/patología , Insuficiencia Renal Crónica/etiología , RoscovitinaRESUMEN
Placental function requires organized growth, transmission of nutrients, and an anti-inflammatory milieu between the maternal and fetal interface, but placental factors important for its function remain unclear. Renalase is a pro-survival, anti-inflammatory flavoprotein found to be critical in other tissues. We examined the potential role of renalase in placental development. PCR, bulk RNA sequencing, immunohistochemistry, and immunofluorescence for renalase and its binding partners, PMCA4b and PZP, were performed on human placental tissue from second-trimester and full-term placentas separated into decidua, placental villi and chorionic plates. Quantification of immunohistochemistry was used to localize renalase across time course from 17 weeks to term. Endogenous production of renalase was examined in placental tissue and organoids. Renalase and its receptor PMCA4b transcripts and proteins were present in all layers of the placenta. Estimated RNLS protein levels did not change with gestation in the decidual samples. However, placental villi contained more renalase immunoreactive cells in fetal than full-term placental samples. RNLS co-labeled with markers for Hofbauer cells and trophoblasts within the placental villi. Endogenous production of RNLS, PMCA4b, and PZP by trophoblasts was validated in placental organoids. Renalase is endogenously expressed throughout placental tissue and specifically within Hofbauer cells and trophoblasts, suggesting a potential role for renalase in placental development and function. Future studies should assess renalase's role in normal and diseased human placenta.
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Placenta , Trofoblastos , Vellosidades Coriónicas/metabolismo , Decidua/metabolismo , Femenino , Humanos , Monoaminooxidasa , Placenta/metabolismo , Placentación , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Embarazo , Trofoblastos/metabolismoRESUMEN
Maintenance of a healthy pregnancy is reliant on a successful balance between the fetal and maternal immune systems. Although the maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23â weeks) human placental villi (PV). Consistent with recent reports in other fetal organs, T cells with memory phenotypes, although rare in abundance, were detected within the PV tissue and vasculature. Moreover, we determined that T cells isolated from PV samples may be more proliferative after T cell receptor stimulation than adult T cells at baseline. Collectively, we identified multiple subtypes of fetal immune cells within the PV and specifically highlight the enhanced proliferative capacity of fetal PV T cells.
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Vellosidades Coriónicas/inmunología , Placenta/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Vellosidades Coriónicas/metabolismo , Femenino , Feto/inmunología , Feto/metabolismo , Citometría de Flujo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Células T de Memoria/citología , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Placenta/citología , Placenta/metabolismo , Embarazo , Segundo Trimestre del Embarazo , Receptores de Superficie Celular/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Análisis de la Célula Individual/métodos , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Congenital hepatic fibrosis (CHF) is a developmental liver disease that is caused by mutations in genes that encode ciliary proteins and is characterized by bile duct dysplasia and portal fibrosis. Recent work has demonstrated that mutations in ANKS6 can cause CHF due to its role in bile duct development. Here, we report a novel ANKS6 mutation, which was identified in an infant presenting with neonatal jaundice due to underlying biliary abnormalities and liver fibrosis. Molecular analysis revealed that ANKS6 liver pathology is associated with the infiltration of inflammatory macrophages to the periportal fibrotic tissue and ductal epithelium. To further investigate the role of macrophages in CHF pathophysiology, we generated a novel liver-specific Anks6 knockout mouse model. The mutant mice develop biliary abnormalities and rapidly progressing periportal fibrosis reminiscent of human CHF. The development of portal fibrosis in Anks6 KO mice coincided with the accumulation of inflammatory monocytes and macrophages in the mutant liver. Gene expression and flow cytometric analysis demonstrated the preponderance of M1- over M2-like macrophages at the onset of fibrosis. A critical role for macrophages in promoting peribiliary fibrosis was demonstrated by depleting the macrophages with clodronate liposomes which effectively reduced inflammatory gene expression and fibrosis, and ameliorated tissue histology and biliary function in Anks6 KO livers. Together, this study demonstrates that macrophages play an important role in the initiation of liver fibrosis in ANKS6-deficient livers and their therapeutic elimination may provide an avenue to mitigate CHF in patients.
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Proteínas Portadoras/metabolismo , Colestasis/patología , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Animales , Modelos Animales de Enfermedad , Expresión Génica/fisiología , Inflamación/metabolismo , Inflamación/patología , Hígado/patología , Cirrosis Hepática/patología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Monocitos/patologíaRESUMEN
Generation of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here we reconstruct a single-cell atlas of the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and expose its molecular blueprint.
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Células Enteroendocrinas/metabolismo , Desarrollo Fetal , Insulina/metabolismo , HumanosRESUMEN
Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/MÏ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/MÏ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/MÏ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.
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Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Enterocolitis Necrotizante/patología , Mucosa Gástrica/patología , Monocitos/patología , Receptores de Superficie Celular , Receptores de IgG , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Vasos Sanguíneos/patología , Estudios de Casos y Controles , Quimiotaxis , Enterocolitis Necrotizante/cirugía , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Lactante , Recién Nacido , Intestino Delgado/irrigación sanguínea , Intestino Delgado/patología , Monocitos/inmunología , Neutropenia/etiología , Neutropenia/patología , Neutrófilos/patología , Fagocitosis/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions, including nutrient metabolism, intestinal barrier integrity, and immune regulation. Recent studies suggest that education of intestinal immunity may be ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial, and whether microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially derived metabolites can be detected in second trimester human intestinal samples. Although we were unable to amplify bacterial DNA from fetal intestines, we report a fetal metabolomic intestinal profile with an abundance of bacterially derived and host-derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial-associated metabolites either come from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that were below our detection threshold.
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Bacterias/metabolismo , Feto/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/metabolismo , Intestinos/fisiología , Metaboloma , Adolescente , Bacterias/genética , Bacterias/aislamiento & purificación , Niño , ADN Bacteriano/análisis , ADN Bacteriano/genética , Femenino , Feto/microbiología , Tracto Gastrointestinal/microbiología , Edad Gestacional , Humanos , Lactante , Recién Nacido , Intestinos/microbiología , MasculinoRESUMEN
ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.