RESUMEN
Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.
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Células Epiteliales/metabolismo , Células Epiteliales/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptosis , Secuencia de Bases , Estudios de Casos y Controles , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Epiteliales/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células HEK293 , Células HT29 , Humanos , Enfermedades Inflamatorias del Intestino/genética , Metotrexato/farmacología , Mutación/genética , Fosforilación/efectos de los fármacos , Polimorfismo de Nucleótido Simple/genética , Piroptosis/efectos de los fármacos , Piroptosis/genética , Reproducibilidad de los Resultados , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Regulación hacia Arriba/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genéticaRESUMEN
Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.
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Intestinos/citología , Intestinos/crecimiento & desarrollo , Análisis de la Célula Individual , Células Endoteliales/citología , Sistema Nervioso Entérico/citología , Feto/embriología , Fibroblastos/citología , Humanos , Inmunidad , Enfermedades Intestinales/congénito , Enfermedades Intestinales/patología , Mucosa Intestinal/crecimiento & desarrollo , Intestinos/irrigación sanguínea , Ligandos , Mesodermo/citología , Neovascularización Fisiológica , Pericitos/citología , Células Madre/citología , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.
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Enfermedades Inflamatorias del Intestino/fisiopatología , Mesodermo/fisiología , Animales , Proliferación Celular , Colitis/genética , Colitis/fisiopatología , Colon/fisiología , Células Epiteliales/metabolismo , Fibroblastos/fisiología , Heterogeneidad Genética , Homeostasis , Humanos , Inflamación , Mucosa Intestinal/inmunología , Mucosa Intestinal/fisiología , Intestinos/inmunología , Intestinos/fisiología , Células Madre Mesenquimatosas/fisiología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos , Pericitos , Células RAW 264.7 , Factores de Transcripción SOXD/fisiología , Análisis de la Célula Individual/métodos , Tromboplastina/fisiología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.
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To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.
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Linfocitos T CD4-Positivos/inmunología , Salmonella paratyphi A/inmunología , Salmonella typhi/inmunología , ADP-Ribosil Ciclasa 1/análisis , Adulto , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Linfocitos T CD4-Positivos/química , Células Clonales , Humanos , Fenotipo , Receptores CCR7/análisis , Fiebre Tifoidea/inmunologíaRESUMEN
Genome-wide association studies in ulcerative colitis point to a role for FcγRIIA, a receptor for IgG. Castro-Dopico et al. (2019) find a profound induction of anti-commensal IgG in the colonic mucosa of UC patients and outline a pathway whereby FcγR activation by IgG triggers IL-1ß production, type 17 immunity, and the exacerbation of inflammation.
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Colitis Ulcerosa , Colitis , Colon , Estudio de Asociación del Genoma Completo , Humanos , Inmunoglobulina G , Inflamación , Mucosa IntestinalRESUMEN
Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.
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Arritmias Cardíacas/metabolismo , Calcitonina/metabolismo , Fibrinógeno/biosíntesis , Atrios Cardíacos/metabolismo , Miocardio/metabolismo , Comunicación Paracrina , Animales , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Fibrilación Atrial , Colágeno Tipo I/metabolismo , Femenino , Fibroblastos/metabolismo , Fibrosis/metabolismo , Fibrosis/patología , Atrios Cardíacos/citología , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Humanos , Masculino , Ratones , Miocardio/citología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Receptores de Calcitonina/metabolismoRESUMEN
The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.
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Colon/citología , Colon/patología , Células Epiteliales/clasificación , Células Epiteliales/citología , Salud , Enfermedades Inflamatorias del Intestino/patología , Canales Iónicos/metabolismo , Animales , Biomarcadores/análisis , Colitis Ulcerosa/genética , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Células Epiteliales/microbiología , Células Epiteliales/patología , Predisposición Genética a la Enfermedad/genética , Células Caliciformes/citología , Células Caliciformes/metabolismo , Células Caliciformes/patología , Humanos , Concentración de Iones de Hidrógeno , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Péptidos Natriuréticos/metabolismo , Proteínas/metabolismo , Análisis de la Célula Individual , Células Madre/citología , Células Madre/metabolismo , Células Madre/patología , Uniones Estrechas/metabolismo , Transcripción Genética , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
T cell recognition of a cognate peptide-major histocompatibility complex (pMHC) presented on the surface of infected or malignant cells is of the utmost importance for mediating robust and long-term immune responses. Accurate predictions of cognate pMHC targets for T cell receptors would greatly facilitate identification of vaccine targets for both pathogenic diseases and personalized cancer immunotherapies. Predicting immunogenic peptides therefore has been at the center of intensive research for the past decades but has proven challenging. Although numerous models have been proposed, performance of these models has not been systematically evaluated and their success rate in predicting epitopes in the context of human pathology has not been measured and compared. In this study, we evaluated the performance of several publicly available models, in identifying immunogenic CD8+ T cell targets in the context of pathogens and cancers. We found that for predicting immunogenic peptides from an emerging virus such as severe acute respiratory syndrome coronavirus 2, none of the models perform substantially better than random or offer considerable improvement beyond HLA ligand prediction. We also observed suboptimal performance for predicting cancer neoantigens. Through investigation of potential factors associated with ill performance of models, we highlight several data- and model-associated issues. In particular, we observed that cross-HLA variation in the distribution of immunogenic and non-immunogenic peptides in the training data of the models seems to substantially confound the predictions. We additionally compared key parameters associated with immunogenicity between pathogenic peptides and cancer neoantigens and observed evidence for differences in the thresholds of binding affinity and stability, which suggested the need to modulate different features in identifying immunogenic pathogen versus cancer peptides. Overall, we demonstrate that accurate and reliable predictions of immunogenic CD8+ T cell targets remain unsolved; thus, we hope our work will guide users and model developers regarding potential pitfalls and unsettled questions in existing immunogenicity predictors.
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COVID-19 , Neoplasias , Linfocitos T CD8-positivos/metabolismo , Simulación por Computador , Epítopos de Linfocito T , Humanos , PéptidosRESUMEN
BACKGROUND: Pregnancy represents a physiological state associated with increased vulnerability to severe outcomes from infectious diseases, both for the pregnant person and developing infant. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic may have important health consequences for pregnant individuals, who may also be more reluctant than nonpregnant people to accept vaccination. METHODS: We sought to estimate the degree to which increased severity of SARS-CoV-2 outcomes can be attributed to pregnancy using a population-based SARS-CoV-2 case file from Ontario, Canada. Because of varying propensity to receive vaccination, and changes in dominant circulating viral strains over time, a time-matched cohort study was performed to evaluate the relative risk of severe illness in pregnant women with SARS-CoV-2 compared to other SARS-CoV-2 infected women of childbearing age (10-49 years old). Risk of severe SARS-CoV-2 outcomes was evaluated in pregnant women and time-matched nonpregnant controls using multivariable conditional logistic regression. RESULTS: Compared with the rest of the population, nonpregnant women of childbearing age had an elevated risk of infection (standardized morbidity ratio, 1.28), whereas risk of infection was reduced among pregnant women (standardized morbidity ratio, 0.43). After adjustment for confounding, pregnant women had a markedly elevated risk of hospitalization (adjusted odds ratio, 4.96; 95% confidence interval, 3.86-6.37) and intensive care unit admission (adjusted odds ratio, 6.58; 95% confidence interval, 3.29-13.18). The relative increase in hospitalization risk associated with pregnancy was greater in women without comorbidities than in those with comorbidities (P for heterogeneity, .004). CONCLUSIONS: Given the safety of SARS-CoV-2 vaccines in pregnancy, risk-benefit calculus strongly favors SARS-CoV-2 vaccination in pregnant women.
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COVID-19 , Complicaciones Infecciosas del Embarazo , Femenino , Embarazo , Humanos , Niño , Adolescente , Adulto Joven , Adulto , Persona de Mediana Edad , Masculino , SARS-CoV-2 , COVID-19/epidemiología , Vacunas contra la COVID-19 , Estudios de Cohortes , Complicaciones Infecciosas del Embarazo/epidemiología , Ontario/epidemiología , Resultado del EmbarazoRESUMEN
BACKGROUND: The rapid development of safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a singular scientific achievement. Confounding due to health-seeking behaviors, circulating variants, and differential testing by vaccination status may bias analyses toward an apparent increase in infection severity following vaccination. METHODS: We used data from the Ontario, Canada, Case and Contact Management Database and a provincial vaccination dataset (COVaxON) to create a time-matched cohort of individuals who were hospitalized with SARS-CoV-2 infection. Vaccinated individuals were matched to up to 5 unvaccinated individuals based on test date. Risk of intensive care unit (ICU) admission and death were evaluated using conditional logistic regression. RESULTS: In 20 064 individuals (3353 vaccinated and 16 711 unvaccinated) hospitalized with infection due to SARS-CoV-2 between 1 January 2021 and 5 January 2022, vaccination with 1, 2, or 3 doses significantly reduced the risk of ICU admission and death. An inverse dose-response relationship was observed between vaccine doses received and both outcomes (adjusted odds ratio [aOR] per additional dose for ICU admission, 0.66; 95% confidence interval [CI], .62 to .71; aOR for death, 0.78; 95% CI, .72 to .84). CONCLUSIONS: We identified decreased virulence of SARS-CoV-2 infections in vaccinated individuals, even when vaccines failed to prevent infection sufficiently severe to cause hospitalization. Even with diminished efficacy of vaccines against infection with novel variants of concern, vaccines remain an important tool for reduction of ICU admission and mortality.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevención & control , Virulencia , Vacunación , Ontario/epidemiologíaRESUMEN
Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.
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Células T Invariantes Asociadas a Mucosa/inmunología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , África del Sur del Sahara , Antibacterianos , Diarrea/microbiología , Diarrea/mortalidad , Humanos , Evasión Inmune/genética , Evasión Inmune/fisiología , Células T Invariantes Asociadas a Mucosa/metabolismo , Infecciones por Salmonella/inmunología , Salmonella typhimurium/patogenicidadRESUMEN
The conditions and extent of cross-protective immunity between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and common-cold human coronaviruses (HCoVs) remain open despite several reports of pre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure. Using a pool of functionally evaluated SARS-CoV-2 peptides, we report a map of 126 immunogenic peptides with high similarity to 285 MHC-presented peptides from at least one HCoV. Employing this map of SARS-CoV-2-non-homologous and homologous immunogenic peptides, we observe several immunogenic peptides with high similarity to human proteins, some of which have been reported to have elevated expression in severe COVID-19 patients. After combining our map with SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls, we show that public repertoires for the majority of convalescent patients are dominated by TCRs cognate to non-homologous SARS-CoV-2 peptides. We find that for a subset of patients, >50% of their public SARS-CoV-2-specific repertoires consist of TCRs cognate to homologous SARS-CoV-2-HCoV peptides. Further analysis suggests that this skewed distribution of TCRs cognate to homologous or non-homologous peptides in COVID-19 patients is likely to be HLA-dependent. Finally, we provide 10 SARS-CoV-2 peptides with known cognate TCRs that are conserved across multiple coronaviruses and are predicted to be recognized by a high proportion of the global population. These findings may have important implications for COVID-19 heterogeneity, vaccine-induced immune responses, and robustness of immunity to SARS-CoV-2 and its variants.
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COVID-19 , SARS-CoV-2 , Linfocitos T CD8-positivos , Reacciones Cruzadas , Epítopos de Linfocito T , Humanos , Péptidos , Receptores de Antígenos de Linfocitos T , Glicoproteína de la Espiga del CoronavirusRESUMEN
We estimated the degree to which language used in the high-profile medical/public health/epidemiology literature implied causality using language linking exposures to outcomes and action recommendations; examined disconnects between language and recommendations; identified the most common linking phrases; and estimated how strongly linking phrases imply causality. We searched for and screened 1,170 articles from 18 high-profile journals (65 per journal) published from 2010-2019. Based on written framing and systematic guidance, 3 reviewers rated the degree of causality implied in abstracts and full text for exposure/outcome linking language and action recommendations. Reviewers rated the causal implication of exposure/outcome linking language as none (no causal implication) in 13.8%, weak in 34.2%, moderate in 33.2%, and strong in 18.7% of abstracts. The implied causality of action recommendations was higher than the implied causality of linking sentences for 44.5% or commensurate for 40.3% of articles. The most common linking word in abstracts was "associate" (45.7%). Reviewers' ratings of linking word roots were highly heterogeneous; over half of reviewers rated "association" as having at least some causal implication. This research undercuts the assumption that avoiding "causal" words leads to clarity of interpretation in medical research.
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Investigación Biomédica , Lenguaje , Humanos , CausalidadRESUMEN
BACKGROUND & AIMS: In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. METHODS: A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS: In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS: BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.
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Proteína Morfogenética Ósea 4/metabolismo , Colitis/metabolismo , Colon/metabolismo , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Regeneración , Animales , Comunicación Autocrina , Proteína Morfogenética Ósea 4/genética , Diferenciación Celular , Proliferación Celular , Colitis/genética , Colitis/patología , Colon/patología , Células Epiteliales/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mucosa Intestinal/patología , Intestino Delgado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/patología , Repitelización , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: The pathogenesis of immune checkpoint inhibitor (ICI)-colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. METHODS: We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. RESULTS: We demonstrate CD8+ tissue resident memory T (TRM) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8+ TRM cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8+ TRM cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti-CTLA-4/PD-1 combination therapy and in anti-PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. CONCLUSIONS: Interferon gamma-producing CD8+ TRM cells are a pathological hallmark of ICI-colitis and a novel target for therapy.
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Linfocitos T CD8-positivos/efectos de los fármacos , Colitis/inducido químicamente , Colon/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Memoria Inmunológica/efectos de los fármacos , Interferón gamma/metabolismo , Células T de Memoria/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/metabolismo , Estudios de Casos y Controles , Colitis/tratamiento farmacológico , Colitis/inmunología , Colitis/metabolismo , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/metabolismo , Colon/inmunología , Colon/metabolismo , Estudios Transversales , Perfilación de la Expresión Génica , Humanos , Estudios Longitudinales , Activación de Linfocitos/efectos de los fármacos , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Fenotipo , Piperidinas/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Estudios Prospectivos , Pirimidinas/uso terapéutico , RNA-Seq , Análisis de la Célula Individual , TranscriptomaRESUMEN
NOD2 is an intracellular sensor that contributes to immune defense and inflammation. Here we investigated whether NOD2 mediates its effects through control of microRNAs (miRNAs). miR-29 expression was upregulated in human dendritic cells (DCs) in response to NOD2 signals, and miR-29 regulated the expression of multiple immune mediators. In particular, miR-29 downregulated interleukin-23 (IL-23) by targeting IL-12p40 directly and IL-23p19 indirectly, likely via reduction of ATF2. DSS-induced colitis was worse in miR-29-deficient mice and was associated with elevated IL-23 and T helper 17 signature cytokines in the intestinal mucosa. Crohn's disease (CD) patient DCs expressing NOD2 polymorphisms failed to induce miR-29 upon pattern recognition receptor stimulation and showed enhanced release of IL-12p40 on exposure to adherent invasive E. coli. Therefore, we suggest that loss of miR-29-mediated immunoregulation in CD DCs might contribute to elevated IL-23 in this disease.
Asunto(s)
Enfermedad de Crohn/inmunología , Células Dendríticas/inmunología , Interleucina-23/metabolismo , MicroARNs/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Factor de Transcripción Activador 2/metabolismo , Animales , Células Cultivadas , Células Dendríticas/metabolismo , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Humanos , Inflamación/inmunología , Subunidad p40 de la Interleucina-12/metabolismo , Mucosa Intestinal/inmunología , Ratones , Ratones Noqueados , MicroARNs/genética , Proteína Adaptadora de Señalización NOD2/genética , Polimorfismo de Nucleótido Simple , Células Th17/inmunologíaRESUMEN
HIV-1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV-1 somehow evades detection by the pattern-recognition receptor (PRR) Toll-like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV-1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV-1 trans-infection of CD4+ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV-1 with TLR8+ early endosomes, triggered a pro-inflammatory response, and inhibited trans-infection of CD4+ T cells. Snapin inhibited TLR8 signaling in the absence of HIV-1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV-1 to promote transmission.