Periodic Reporting for period 1 - GLIAMAC (Uncovering Enteric GLIA-MACrophage communication in the intestinal homeostasis and inflammation)
Reporting period: 2018-12-01 to 2020-11-30
It is well known that intestinal myeloid cells play a role in intestinal homeostasis and inflammation, in addition to that their phenotype and function can be regulated by a number of factors. We believe that glia-derived molecules help maintaining intestinal homeostasis via regulation of myeloid cells, our aim at demonstrating that a dysregulated glia-immune cell crosstalk leads to the development of intestinal inflammation. Therefore, for the first time, GLIAMAC will investigate the molecules and pathways involved in glia-immune interaction and elucidate the role of Enteric Glial Cells (EGCs) in the pathogenesis of intestinal inflammation. Based on my preliminary data in the current project (GLIAMAC), I will test the hypothesis that EGCs plays a central role in “educating” intestinal myeloid cells and promoting immunological tolerance. Using novel multicellular culture approaches and glial-specific transgenic mouse models, I will define the molecules and the molecular pathways involved in the enteric glia-myeloid cell crosstalk and define the relevance of this neuro-immune interaction in the course of intestinal inflammation. I am convinced that identification of new molecules and pathways involved in enteric glia-immune cell crosstalk will represent a major breakthrough in elucidating the pathogenesis of intestinal immune-mediated diseases such as Inflammatory Bowel Disease (IBD) or post-operative ileus (POI) and will potentially give rise to a new class of molecules to treat and favour remission in patients affected by intestinal inflammation. This knowledge may give rise to novel therapeutic approaches to treat patients affected by chronic intestinal inflammatory disorders. Moreover, the knowledge about the impact of neurological signals on immune cells and homeostasis can be extended beyond the context of the intestinal mucosal homeostasis and may be valuable in the context of several acute and chronic inflammatory conditions and autoimmune diseases.
Based on my preliminary data in the current project (GLIAMAC),We test the hypothesis that enteric glia play a central role in “educating” intestinal myeloid cells and promoting immunological tolerance.
Briefly, the gastrointestinal tract (GI) constitutes the largest area of body contact with the external environment. In order to control microbial aggressions and to maintain the homeostasis of the gut, the innate immune system, the enteric nervous system and intestinal epithelium facilitated the cohabitation of beneficial microorganisms for the establishment of regulatory networks that prevent inflammation such as Inflammatory Bowel Disease (IBD) and post-operative ileus (POI).
The intestinal immune and nervous systems sense and integrate luminal cues and regulate physiological processes, including GI motility1,2. In the gut wall, the ENS forms clusters of neurons surrounded by Enteric Glial Cells (EGCs) in a network of ganglia. Historically, EGCs were mainly considered only as supporting cells of enteric neurons. However, recent evidences suggest that enteric glia have a much border participation in gastrointestinal physiology, contributing to motility, preserving epithelial barrier integrity and regulating the innate inflammatory response in the gut.
In the enteric muscularis externa (ME) the resident Macrophages (Mφs) population, which closely interact with the enteric nervous system (ENS), regulate intestinal functioning via the secretion of bone morphogenetic protein 2 (BMP2), which supports the function and differentiation of enteric neurons in the absence of infections. Furthermore, evidence suggests that intestinal-resident macrophage populations upon tissue damage detect damage-associated and pathogen-associated molecular patterns and attract circulating monocytes and neutrophils. Moreover, the role of EGCs during tissue damage it is unknown, in our preliminary data showed that EGCs are in close proximity with the intestinal Mφs. Thus, how EGCs and myeloid cells communicate under tissue damage remains still completely unknown.
Based on my preliminary data in the current project (GLIAMAC), I test the hypothesis that enteric glia play a central role in “educating” intestinal myeloid cells and promoting immunological tolerance. Using novel emergence techniques including single cell RNA sequencing (scRNA-seq) help us to uncover the molecular mechanisms driving the differentiation of monocytes towards pro-resolving Mφs as we can evaluate Mφs in different states during this process. Therefore, we will employ scRNA-seq to decipher the cues and the transcription factors driving the differentiation into pro-resolving Mφs in a model of small intestinal inflammation. Additionally, we evaluated how Mφs are influenced by their environment in the ME, where they are close of the EGCs. Thus, we analyze how EGCs attract circulating monocytes and promoted the differentiation of pro-resolving Mφs to support the resolution of inflammation.
Based on my preliminary data in the current project (GLIAMAC),We test the hypothesis that enteric glia play a central role in “educating” intestinal myeloid cells and promoting immunological tolerance.
Briefly, the gastrointestinal tract (GI) constitutes the largest area of body contact with the external environment. In order to control microbial aggressions and to maintain the homeostasis of the gut, the innate immune system, the enteric nervous system and intestinal epithelium facilitated the cohabitation of beneficial microorganisms for the establishment of regulatory networks that prevent inflammation such as Inflammatory Bowel Disease (IBD) and post-operative ileus (POI).
The intestinal immune and nervous systems sense and integrate luminal cues and regulate physiological processes, including GI motility1,2. In the gut wall, the ENS forms clusters of neurons surrounded by Enteric Glial Cells (EGCs) in a network of ganglia. Historically, EGCs were mainly considered only as supporting cells of enteric neurons. However, recent evidences suggest that enteric glia have a much border participation in gastrointestinal physiology, contributing to motility, preserving epithelial barrier integrity and regulating the innate inflammatory response in the gut.
In the enteric muscularis externa (ME) the resident Macrophages (Mφs) population, which closely interact with the enteric nervous system (ENS), regulate intestinal functioning via the secretion of bone morphogenetic protein 2 (BMP2), which supports the function and differentiation of enteric neurons in the absence of infections. Furthermore, evidence suggests that intestinal-resident macrophage populations upon tissue damage detect damage-associated and pathogen-associated molecular patterns and attract circulating monocytes and neutrophils. Moreover, the role of EGCs during tissue damage it is unknown, in our preliminary data showed that EGCs are in close proximity with the intestinal Mφs. Thus, how EGCs and myeloid cells communicate under tissue damage remains still completely unknown.
Based on my preliminary data in the current project (GLIAMAC), I test the hypothesis that enteric glia play a central role in “educating” intestinal myeloid cells and promoting immunological tolerance. Using novel emergence techniques including single cell RNA sequencing (scRNA-seq) help us to uncover the molecular mechanisms driving the differentiation of monocytes towards pro-resolving Mφs as we can evaluate Mφs in different states during this process. Therefore, we will employ scRNA-seq to decipher the cues and the transcription factors driving the differentiation into pro-resolving Mφs in a model of small intestinal inflammation. Additionally, we evaluated how Mφs are influenced by their environment in the ME, where they are close of the EGCs. Thus, we analyze how EGCs attract circulating monocytes and promoted the differentiation of pro-resolving Mφs to support the resolution of inflammation.
1.CD45+ immune cells in healthy and inflamed muscularis by ScRNA-seq analysis.
Our results indicate that during muscularis inflammation increase the recruitment of not only macrophages if not other immune cells, which can be critical regulators for the resolution of the inflammation.
2.Monocytes/Mφs in muscularis externa during healthy and intestinal inflammation.
We found that there are two major population monocyte-to-Mφ differentiation pathways in the inflamed muscularis giving rise to an Igf1+ and Timp2+ Mφ population during the resolution of MI.
3.Recruitment of Cx3cr1gfp/+ Mφs in muscularis externa during inflammation
We show that majority of the subpopulations of Mφ could be detected by FACS analysis. On the other hand, phenotype the CCR2-/- and which subpopulation of Mφ has important phenotype to resolve the inflammation.
4. Glial analysis in the muscularis externa after IM.
We detected an increase production of Ccl2 and Csf1 by picked ganglia from WT mice ME in the early time points after IM and by the analysis of RNA of EGC, meaning that EGC sense the inflammatory environment and respond faster to the recruitment of monocytes to help repairing the tissue damage.
5.Monocytes sense the EGC environment which orchestrated their fitness.
Our data show in vitro and ex vivo how an environment of EGC derived ligands can educate enteric monocytes to anti-inflammatory Mφ to induce the homeostasis in the pathologic tissue.
Our results indicate that during muscularis inflammation increase the recruitment of not only macrophages if not other immune cells, which can be critical regulators for the resolution of the inflammation.
2.Monocytes/Mφs in muscularis externa during healthy and intestinal inflammation.
We found that there are two major population monocyte-to-Mφ differentiation pathways in the inflamed muscularis giving rise to an Igf1+ and Timp2+ Mφ population during the resolution of MI.
3.Recruitment of Cx3cr1gfp/+ Mφs in muscularis externa during inflammation
We show that majority of the subpopulations of Mφ could be detected by FACS analysis. On the other hand, phenotype the CCR2-/- and which subpopulation of Mφ has important phenotype to resolve the inflammation.
4. Glial analysis in the muscularis externa after IM.
We detected an increase production of Ccl2 and Csf1 by picked ganglia from WT mice ME in the early time points after IM and by the analysis of RNA of EGC, meaning that EGC sense the inflammatory environment and respond faster to the recruitment of monocytes to help repairing the tissue damage.
5.Monocytes sense the EGC environment which orchestrated their fitness.
Our data show in vitro and ex vivo how an environment of EGC derived ligands can educate enteric monocytes to anti-inflammatory Mφ to induce the homeostasis in the pathologic tissue.
Our results identified that EGCs can control tissue-protection by stimulating the differentiation of enteric monocytes to pro-resolving Mφ. We identified that tissue damage in the gut results in early secretion of CCL2 by EGCs leading to the recruitment of monocytes, after which differentiate to pro-resolving Mφ by other factors secreted by EGCs such as CSF-1. Our data uncover EGCs as a novel therapeutic target, which can secrete tissue-protective cytokines in pathology conditions. Additional experiments in vivo to explore the role of EGCs and characterize in vivo the EGC derived factors orchestrated the education of Mφs are still needed. In summary, we revealed a largely unexplored cells and derived glial factors that control monocytes recruitment and fitness; thus, opening new horizons for our futures innovative research on EGC- Mφ in enteric inflammatory pathologies.