RESUMEN
Fibroblasts are ubiquitous cells that can adopt many functional states. As tissue-resident sentinels, they respond to acute damage signals and shape the earliest events in fibrotic and immune-mediated inflammatory diseases. Upon sensing an insult, fibroblasts produce chemokines and growth factors to organize and support the response. Depending on the size and composition of the resulting infiltrate, these activated fibroblasts may also begin to contract or relax thus changing local stiffness within the tissue. These early events likely contribute to the divergent clinical manifestations of fibrotic and immune-mediated inflammatory diseases. Further, distinct changes to the cellular composition and signaling dialogue in these diseases drive progressive fibroblasts specialization. In fibrotic diseases, fibroblasts support the survival, activation and differentiation of myeloid cells, granulocytes and innate lymphocytes, and produce most of the pathogenic extracellular matrix proteins. Whereas, in immune-mediated inflammatory diseases, sequential accumulation of dendritic cells, T cells and B cells programs fibroblasts to support local, destructive adaptive immune responses. Fibroblast specialization has clear implications for the development of effective induction and maintenance therapies for patients with these clinically distinct diseases.
Asunto(s)
Fibroblastos , Fibrosis , Inflamación , Humanos , Fibroblastos/inmunología , Fibroblastos/patología , Inflamación/inmunología , Inflamación/patología , AnimalesRESUMEN
BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.
Asunto(s)
Colitis , Enfermedad de Crohn , Animales , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Constricción Patológica , Intestinos/patología , Colitis/patología , Fibroblastos/patologíaRESUMEN
Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos , Fibrosis Pulmonar , Humanos , Ratones , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Interleucina-17/metabolismo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Cicatriz , Macrófagos/patología , Inflamación/patología , Proteínas de Unión a Ácidos Grasos/metabolismo , Glicoproteínas de Membrana , Receptores InmunológicosRESUMEN
Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.
RESUMEN
The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.
Asunto(s)
Leucocitos/patología , Microambiente Tumoral/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Automatización , Calor , Humanos , Procesamiento de Imagen Asistido por Computador , Adhesión en Parafina , Coloración y Etiquetado , Células del Estroma/metabolismo , Fijación del TejidoRESUMEN
The type 3 cytokines IL-17 and IL-22 play a crucial, well synchronized physiological role in wound healing and repairing tissue damage due to infections or injury at barrier surfaces. These cytokines act on epithelial cells to induce secretion of early immune mediators, recruitment of inflammatory cells to the site of injury, and to trigger tissue repair mechanisms. However, if the damage persists or if these cytokines are dysregulated, then they contribute to a number of inflammatory pathologies, autoimmune conditions and cancer. The liver is a multifunctional organ that plays an essential role in metabolism, detoxification, and immune surveillance. It is also exposed to a variety of pathogens, toxins and injuries. Over the past decade, IL-17 and IL-22 have been implicated in various aspects of liver inflammation. IL-17 is upregulated in chronic liver injury and associated with liver disease progression. In contrast, IL-22 was shown to be hepatoprotective during acute liver injury but exhibited inflammatory effects in other models. Furthermore, IL-22 and IL-17 are both associated with poor prognosis in liver cancer. Finally, the regulatory mechanisms governing the physiological versus the pathological role of these two cytokines during acute and chronic liver injury remain poorly understood. In this review, we will summarize the current state of knowledge about IL-17 and IL-22 in wound healing during acute and chronic liver injury, their contribution to pathogenesis, their regulation, and their role in the transition from advanced liver disease to liver cancer.
Asunto(s)
Carcinoma Hepatocelular/inmunología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Interleucina-17/metabolismo , Interleucinas/metabolismo , Cirrosis Hepática/inmunología , Neoplasias Hepáticas/inmunología , Animales , Carcinoma Hepatocelular/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/microbiología , Enfermedad Hepática Inducida por Sustancias y Drogas/virología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/microbiología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/virología , Humanos , Inmunidad Innata , Neoplasias Hepáticas/patología , Interleucina-22RESUMEN
Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (Tregs), but its contribution to liver fibrosis is still poorly understood. Here, we evaluated the contribution of type 3 inflammation in liver fibrosis using clinical liver biopsies, in vitro stimulation of primary HSCs, and in vivo mouse models. We report dysregulated type 3 responses in fibrotic lesions with increased IL-17+CD4+/FOXP3hiCD4+ ratio and increased IL-17 and IL-22 production in advanced liver fibrosis. Neutrophils and mast cells were the main sources of IL-17 in situ in humans. In addition, we demonstrate a new profibrotic function of IL-22 through enhancement of transforming growth factor-ß signaling in HSCs in a p38 mitogen-activated protein kinase-dependent manner. In vivo, IL-22RA1 knockout mice exhibited reduced fibrosis in response to thioacetamide and carbon tetrachloride. Blocking either IL-22 or IL-17 production using aryl hydrocarbon receptor or RAR-related orphan receptor gamma-t antagonists resulted in reduced fibrosis. Together, these data have identified a pathogenic role for type 3 immune response mediated by IL-22 in driving liver fibrosis during chronic liver injury.
Asunto(s)
Interleucina-17/inmunología , Interleucinas/inmunología , Cirrosis Hepática/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Compuestos Azo/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Interleucina-17/antagonistas & inhibidores , Interleucina-17/metabolismo , Interleucinas/antagonistas & inhibidores , Interleucinas/metabolismo , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazoles/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Interleucina-22RESUMEN
Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in the liver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dysfunction, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immune responses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4-deficient mice (type 1-polarized) were highly resistant to NASH. This protection was associated with an increased hepatic interferon-γ (IFN-γ) signature. Conversely, IFN-γ-deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor-ß (TGF-ß) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen in expanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflammation in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-ß and IL-13 signaling attenuated the fibrotic machinery more completely than TGF-ß alone in NAFLD-associated fibrosis. Thus, although type 2 immunity maintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaboratively with TGF-ß in the liver.
Asunto(s)
Progresión de la Enfermedad , Inmunidad , Enfermedades Metabólicas/inmunología , Enfermedades Metabólicas/prevención & control , Enfermedad del Hígado Graso no Alcohólico/inmunología , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Factor de Crecimiento Transformador beta/metabolismo , Tejido Adiposo/patología , Animales , Dieta Alta en Grasa , Eosinófilos/patología , Humanos , Inflamación/patología , Interferón gamma/deficiencia , Interferón gamma/metabolismo , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/patologíaRESUMEN
HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood, a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly ß and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts, increased viral load, lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target.
Asunto(s)
Biomarcadores/sangre , Infecciones por VIH/sangre , Mediadores de Inflamación/sangre , Interleucinas/sangre , Adulto , Recuento de Linfocito CD4 , Relación CD4-CD8 , Células Cultivadas , Estudios de Cohortes , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica/métodos , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Interleucinas/genética , Receptores de Lipopolisacáridos/sangre , Receptores de Lipopolisacáridos/genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Carga Viral/inmunologíaRESUMEN
Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis, characterized by enhanced extracellular matrix production and altered degradation. Activation of HSCs can be modulated by cytokines produced by immune cells. Recent reports have implicated the proinflammatory cytokine IL-17A in liver fibrosis progression. We hypothesized that IL-17A may enhance activation of HSCs and induction of the fibrogenic signals in these cells. The human HSC line LX2 and primary human HSCs were stimulated with increasing doses of IL-17A and compared with TGF-ß- and PBS-treated cells as positive and negative controls, respectively. IL-17A alone did not induce activation of HSCs. However, IL-17A sensitized HSCs to the action of suboptimal doses of TGF-ß as confirmed by strong induction of α-smooth muscle actin, collagen type I (COL1A1), and tissue inhibitor of matrix metalloproteinase I gene expression and protein production. IL-17A specifically upregulated the cell surface expression of TGF-ßRII following stimulation. Pretreatment of HSCs with IL-17A enhanced signaling through TGF-ßRII as observed by increased phosphorylation of SMAD2/3 in response to stimulation with suboptimal doses of TGF-ß. This enhanced TGF-ß response of HSCs induced by IL-17A was JNK-dependent. Our results suggest a novel profibrotic function for IL-17A by enhancing the response of HSCs to TGF-ß through activation of the JNK pathway. IL-17A acts through upregulation and stabilization of TGF-ßRII, leading to increased SMAD2/3 signaling. These findings represent a novel example of cooperative signaling between an immune cytokine and a fibrogenic receptor.
Asunto(s)
Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Interleucina-17/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Cirrosis Hepática/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/farmacología , Actinas/biosíntesis , Línea Celular , Colágeno Tipo I/biosíntesis , Cadena alfa 1 del Colágeno Tipo I , Células Estrelladas Hepáticas/patología , Humanos , Interleucina-17/farmacología , Hígado/citología , Hígado/patología , Cirrosis Hepática/inmunología , Fosforilación , Proteínas Serina-Treonina Quinasas/biosíntesis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Transducción de Señal/inmunología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Regulación hacia ArribaRESUMEN
Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). In vitro supplementation of Tim-3(high) HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs.
Asunto(s)
Proliferación Celular , Galectinas/inmunología , Hepatitis C/inmunología , Interleucinas/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Enfermedad Aguda , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Células Cultivadas , Femenino , Estudios de Seguimiento , Galectinas/biosíntesis , Regulación de la Expresión Génica/inmunología , Receptor 2 Celular del Virus de la Hepatitis A , Hepatitis C/sangre , Hepatitis C/patología , Humanos , Interleucinas/sangre , Masculino , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Células Th17/metabolismo , Células Th17/patologíaRESUMEN
Single nucleotide polymorphisms (SNPs) in the proximity of the interleukin-28B (IL28B) gene can predict spontaneous resolution of hepatitis C virus (HCV) infection and response to interferon therapy. Screening for this polymorphism has become part of the standard criteria for the management of HCV-infected patients, hence the need for a rapid, cost-effective screening method. Here, we describe a rapid PCR-based test to screen for two IL28B SNPs (rs12979860 and rs8099917). We used this test to investigate IL28B polymorphism and prevalence in a cohort of French Canadian injection drug users who are part of a unique population known to have a strong genetic founder effect. This population had lower linkage disequilibrium between the two tested SNPs as compared to other cohorts (|d'| = 0.68, r = 0.59). The special genetic makeup should be considered in the management of HCV-infected patients within that population.
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Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/diagnóstico , Hepatitis C Crónica/genética , Interleucinas/genética , Reacción en Cadena de la Polimerasa/métodos , Adulto , Canadá/epidemiología , Estudios de Cohortes , Femenino , Genotipo , Hepatitis C Crónica/epidemiología , Humanos , Interferones , Desequilibrio de Ligamiento , Masculino , Polimorfismo de Nucleótido Simple , PrevalenciaRESUMEN
Immunoimaging scanning probe microscopy was utilized for the low-level detection and quantification of biotinylated G4 poly(amidoamine) PAMAM dendrimers. Results were compared to those of high-performance liquid chromatography (HPLC) and found to provide a vastly improved analytical method for the low-level detection of dendrimers, improving the limit of detection by a factor of 1000 (LOD = 2.5 × 10(-13) moles). The biorecognition method is reproducible and shows high specificity and good accuracy. In addition, the capture assay platform shows a promising approach to patterning dendrimers for nanotechnology applications.