Dissecting the cellular basis of Inflammatory Bowel Disease through Single-cell RNA sequencing

[eng] Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases that show perplexing heterogeneity which is poorly understood at a cellular level. We applied single-cell RNA sequencing and CosMxTM Spatial Molecular Imaging to human colon and observed profound changes in the epithelium of IBD patients with the presence of cell loss and the increase of markers linked to inflammation. Additionally, we found the highest diversity among patients in the myeloid and stromal compartment. We characterized for the first time the transcriptional signatures of intestinal neutrophils finding three different neutrophil states, including a CXCR4+ population not found in blood. We detected, in both healthy and inflamed colon, a novel resident macrophage population (M0), that differs from the previously described M2. In addition, we found uniquely in patients a variety of classical inflammatory M1 macrophages and a previously undescribed population that we called Inflammation-Dependent Alternative (IDA) macrophages. Single-cell transcriptomic, spatial data and immunostaining uncovered two distinct populations of IDA macrophages: subepithelial IDA macrophages highly expressing NRG1 that can act on the epithelium; and NRG1low IDA macrophages, which were expanded within the submucosa and in Crohn’s disease granulomas. NRG1 was found to be highly upregulated in IBD, especially UC. Remarkably, we also detected expression of NRG1 in peri-cryptal S2b fibroblasts in healthy tissue and in other fibroblast populations in IBD. Additionally, S2b fibroblasts express the neuromodulator NPY in healthy samples and was profoundly downregulated in IBD patients. Lamina propria S1 fibroblasts decrease in IBD but remaining cells show an increase in the expression of pro- inflammatory genes. Thus, the peri-cryptal and S1 fibroblasts suffer a rewiring in IBD. We also identified a population of inflammatory fibroblasts in IBD which had a unique transcriptomic profile. Remarkably, we observed high co-localization of inflammatory fibroblasts and novel IDA macrophages through CosMx SMI, suggesting cellular crosstalk. The combination of single-cell sequencing and spatial analysis allowed us to unravel the complexity of cell populations in inflammatory bowel disease and find potential cross- talks between cell types that could help in understanding the disease and identifying new therapeutic targets.[spa] La enfermedad de Crohn (EC) y la colitis ulcerosa (CU) son enfermedades crónicas inflamatorias del intestino (EEI) que muestran una heterogeneidad desconcertante que no está bien descrita a nivel celular. En este estudio hemos aplicado secuenciación de ARN de célula única y CosMxTM Spatial Molecular Imaging al colon humano y hemos observado cambios profundos en el epitelio de pacientes con EII con la presencia de pérdida celular y el aumento de marcadores vinculados a la inflamación. Además, encontramos la mayor diversidad entre pacientes en el compartimento mieloide y estromal. Caracterizamos por primera vez las firmas transcripcionales de los neutrófilos intestinales, encontrando tres estados diferentes de neutrófilos, incluida una población CXCR4+ no encontrada en sangre. Detectamos, tanto en el colon sano como inflamado, una nueva población de macrófagos residentes (M0), que difiere de la M2 descrita previamente. Además, encontramos de manera única en pacientes una variedad de macrófagos inflamatorios clásicos M1 y una población previamente no descrita que llamamos macrófagos alternativos dependientes de la inflamación (IDA). Los datos transcriptómicos de células únicas, espaciales y de inmunotinción nos permitieron descubrir dos poblaciones distintas de macrófagos IDA: macrófagos IDA subepiteliales altamente expresando NRG1 que pueden actuar sobre el epitelio; y macrófagos IDA con expresión baja de NRG1, que se expanden dentro de la submucosa y en los granulomas de un paciente de EC. Observamos que NRG1 estaba altamente regulado en la EII, especialmente en la CU. Sorprendentemente, también detectamos la expresión de NRG1 en fibroblastos S2b peri- criptales en tejido sano y en otras poblaciones de fibroblastos en EII. Además, los fibroblastos S2b expresan el neuromodulador NPY en muestras sanas y se redujeron profundamente en pacientes con EII. Los fibroblastos S1 de la lámina propia disminuyen en la EII pero las células restantes muestran un aumento en la expresión de genes proinflamatorios. Así, los fibroblastos peri-criptales y S1 sufren un recableado en la EII. También identificamos una población de fibroblastos inflamatorios en la EII que tenía un perfil transcriptómico único. Sorprendentemente, observamos una alta co-localización de fibroblastos inflamatorios y nuevos macrófagos IDA a través de CosMx SMI, lo que sugiere una diafonía celular. La combinación de secuenciación unicelular y análisis espacial nos permitió desentrañar la complejidad de las poblaciones celulares en la enfermedad inflamatoria intestinal y encontrar posibles interacciones cruzadas entre tipos celulares que podrían ayudar a comprender la enfermedad e identificar nuevos objetivos terapéuticos

Dissecting the cellular basis of Inflammatory Bowel Disease through Single-cell RNA sequencing

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS)[eng] Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases that show perplexing heterogeneity which is poorly understood at a cellular level. We applied single-cell RNA sequencing and CosMxTM Spatial Molecular Imaging to human colon and observed profound changes in the epithelium of IBD patients with the presence of cell loss and the increase of markers linked to inflammation. Additionally, we found the highest diversity among patients in the myeloid and stromal compartment. We characterized for the first time the transcriptional signatures of intestinal neutrophils finding three different neutrophil states, including a CXCR4+ population not found in blood. We detected, in both healthy and inflamed colon, a novel resident macrophage population (M0), that differs from the previously described M2. In addition, we found uniquely in patients a variety of classical inflammatory M1 macrophages and a previously undescribed population that we called Inflammation-Dependent Alternative (IDA) macrophages. Single-cell transcriptomic, spatial data and immunostaining uncovered two distinct populations of IDA macrophages: subepithelial IDA macrophages highly expressing NRG1 that can act on the epithelium; and NRG1low IDA macrophages, which were expanded within the submucosa and in Crohn’s disease granulomas. NRG1 was found to be highly upregulated in IBD, especially UC. Remarkably, we also detected expression of NRG1 in peri-cryptal S2b fibroblasts in healthy tissue and in other fibroblast populations in IBD. Additionally, S2b fibroblasts express the neuromodulator NPY in healthy samples and was profoundly downregulated in IBD patients. Lamina propria S1 fibroblasts decrease in IBD but remaining cells show an increase in the expression of pro- inflammatory genes. Thus, the peri-cryptal and S1 fibroblasts suffer a rewiring in IBD. We also identified a population of inflammatory fibroblasts in IBD which had a unique transcriptomic profile. Remarkably, we observed high co-localization of inflammatory fibroblasts and novel IDA macrophages through CosMx SMI, suggesting cellular crosstalk. The combination of single-cell sequencing and spatial analysis allowed us to unravel the complexity of cell populations in inflammatory bowel disease and find potential cross- talks between cell types that could help in understanding the disease and identifying new therapeutic targets.[spa] La enfermedad de Crohn (EC) y la colitis ulcerosa (CU) son enfermedades crónicas inflamatorias del intestino (EEI) que muestran una heterogeneidad desconcertante que no está bien descrita a nivel celular. En este estudio hemos aplicado secuenciación de ARN de célula única y CosMxTM Spatial Molecular Imaging al colon humano y hemos observado cambios profundos en el epitelio de pacientes con EII con la presencia de pérdida celular y el aumento de marcadores vinculados a la inflamación. Además, encontramos la mayor diversidad entre pacientes en el compartimento mieloide y estromal. Caracterizamos por primera vez las firmas transcripcionales de los neutrófilos intestinales, encontrando tres estados diferentes de neutrófilos, incluida una población CXCR4+ no encontrada en sangre. Detectamos, tanto en el colon sano como inflamado, una nueva población de macrófagos residentes (M0), que difiere de la M2 descrita previamente. Además, encontramos de manera única en pacientes una variedad de macrófagos inflamatorios clásicos M1 y una población previamente no descrita que llamamos macrófagos alternativos dependientes de la inflamación (IDA). Los datos transcriptómicos de células únicas, espaciales y de inmunotinción nos permitieron descubrir dos poblaciones distintas de macrófagos IDA: macrófagos IDA subepiteliales altamente expresando NRG1 que pueden actuar sobre el epitelio; y macrófagos IDA con expresión baja de NRG1, que se expanden dentro de la submucosa y en los granulomas de un paciente de EC. Observamos que NRG1 estaba altamente regulado en la EII, especialmente en la CU. Sorprendentemente, también detectamos la expresión de NRG1 en fibroblastos S2b peri- criptales en tejido sano y en otras poblaciones de fibroblastos en EII. Además, los fibroblastos S2b expresan el neuromodulador NPY en muestras sanas y se redujeron profundamente en pacientes con EII. Los fibroblastos S1 de la lámina propia disminuyen en la EII pero las células restantes muestran un aumento en la expresión de genes proinflamatorios. Así, los fibroblastos peri-criptales y S1 sufren un recableado en la EII. También identificamos una población de fibroblastos inflamatorios en la EII que tenía un perfil transcriptómico único. Sorprendentemente, observamos una alta co-localización de fibroblastos inflamatorios y nuevos macrófagos IDA a través de CosMx SMI, lo que sugiere una diafonía celular. La combinación de secuenciación unicelular y análisis espacial nos permitió desentrañar la complejidad de las poblaciones celulares en la enfermedad inflamatoria intestinal y encontrar posibles interacciones cruzadas entre tipos celulares que podrían ayudar a comprender la enfermedad e identificar nuevos objetivos terapéuticos

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in human inflammatory bowel disease

Ulcerative colitis and Crohn's disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity.This work was funded by grant PID2021-123918OB-I00 from MCIN/AEI/ 10.13039/501100011033 and co-funded by “FEDER A way to make Europe”. AM-C, VG and ID were funded by grant Grant #2008-04050 from The Leona and Harry B. Helmsley Charitable Trust. EM-A is funded by grant RH042155 (RTI2018-096946-B-I00) from Ministerio de Ciencia e Innovacion. IA-T is funded by grant 831434-2 (CE_IMI2-2018-14 call). HH received support for the project PID2020-115439GB-I00- funded by MCIN/AEI/10.13039/501100011033. This publication is part of a project that has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 831434. This project also has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 848028

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in human inflammatory bowel disease

Ulcerative colitis and Crohn's disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity

Dissecting common and unique effects of anti-alpha4beta7 and anti-tumor necrosis factor treatment in ulcerative colitis

Background and Aims:Vedolizumab is an anti-α4β7 antibody approved for the treatment of ulcerative colitis [UC]. Although it is assumed that vedolizumab blocks intestinal homing of lymphocytes, its effects on different intestinal cell populations are not fully stablished. In order to establish the unique mechanisms of action of vedolizumab in UC patients, we compared its effects to those induced by anti-tumour necrosis factor [TNF]. Methods:patients with active UC [endoscopic Mayo score >1] starting vedolizumab [n = 33] or anti-TNF [n = 45] and controls [n = 22] were included. Colon biopsies [at weeks 0, 14 and 46] and blood samples [at weeks 0, 2, 6, 14, 30 and 46] were used for cell phenotyping, transcriptional analysis [qPCR], and to measure receptor occupancy. Results:Vedolizumab, in contrast to anti-TNF, significantly reduced the proportion of α4β7+ cells within intestinal T subsets while preserving the percentage of α4β7+ plasma cells. The marked decrease in α4β7 did not change the percentage of colonic αEβ7+ cells [at 46 weeks]. Both vedolizumab and anti-TNF significantly downregulated inflammation-related genes in the colon of responders [Mayo score < 2]. Moreover, both treatments significantly decreased the percentage of intestinal, but not blood, total lymphocytes [T and plasma cells], as well as the proportion of α4β1+ cells within intestinal T lymphocytes. Conclusions:Our data show that while vedolizumab and anti-TNF block two unrelated targets, they induce remarkably similar effects. On the other hand, vedolizumab's unique mechanism of action relies on blocking intestinal trafficking of α4β7 T cells, despite effectively binding to B and plasma cells that express α4β7

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in human inflammatory bowel disease

Abstract Ulcerative colitis and Crohn’s disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity