Periodic Reporting for period 1 - ILCSIGNALLING (Deciphering cell-cell and cell-microbiome interactions of innate lymphoid cells at the single cell level)
Okres sprawozdawczy: 2018-01-01 do 2019-12-31
The gastrointestinal tract encompasses a large and diverse microbial ecosystem, termed the gut microbiome which extensively interacts with gut immune system, ensuring tolerance in times of ‘peace’ yet potently reacting against invading pathogens. Recently discovered innate lymphoid cells (ILCs) appear to be a key player in the immune network of the gut, as they function in regulation of homeostasis between gut resident commensal microbiota and immune system, which involves connecting innate and adaptive immunity. Yet, the heterogeneity of this cell compartment is not fully understood as well as the extent of cell-to-cell interactions involved in these process. Similarly, in the other key organ of gastrointestinal system, the liver, the extent of interaction of cells and the impact of the microbiota is not clear, both in health and in disease, such as acute liver failure. Treatment of ALF is limited, mainly consisting of supportive care and liver transplantation.
The key objective of this project is to develop a quantitative single-cell transcriptomics-based analysis method to gain comprehensive describe cells of the gastrointestinal system in health and disease, understanding of cell-to-cell communication network in the immune system in health and disease and decipher the impact of the microbiota. This is important, as dysregulation of the immune system of the gut leads to inflammatory diseases such as IBD, more severe outcomes of gastrointestinal infections and more severe outcomes of liver diseases. The objective of this study is to understand cellular and molecular mechanisms driving interactions between cells under stress in the disease model. We identified population of ILCs that are microbiota dependent, in absence of microbiota the number of ILC1 dramatically decreases, and these cells are replaced by ILC3p subpopulation. This leads to skewing the signalling balance in the liver, as ILC1 are producers of key soluble factors such as interferon gamma nad Ccl5. We also deeply characterised the process and found molecular mechanisms driving infiltration of monocytes to the liver upon liver damage. We observed amelioration of the acute liver failure in mouse model targeting TLR signalling, MAPK pathway, Myc transcription factor and microbiota, suggesting that these targets we have identified may indeed be clinically relevant. Hence, we demonstrated that a detailed ALF cellular decoding may enable pathway-specific therapeutic intervention.
The key objective of this project is to develop a quantitative single-cell transcriptomics-based analysis method to gain comprehensive describe cells of the gastrointestinal system in health and disease, understanding of cell-to-cell communication network in the immune system in health and disease and decipher the impact of the microbiota. This is important, as dysregulation of the immune system of the gut leads to inflammatory diseases such as IBD, more severe outcomes of gastrointestinal infections and more severe outcomes of liver diseases. The objective of this study is to understand cellular and molecular mechanisms driving interactions between cells under stress in the disease model. We identified population of ILCs that are microbiota dependent, in absence of microbiota the number of ILC1 dramatically decreases, and these cells are replaced by ILC3p subpopulation. This leads to skewing the signalling balance in the liver, as ILC1 are producers of key soluble factors such as interferon gamma nad Ccl5. We also deeply characterised the process and found molecular mechanisms driving infiltration of monocytes to the liver upon liver damage. We observed amelioration of the acute liver failure in mouse model targeting TLR signalling, MAPK pathway, Myc transcription factor and microbiota, suggesting that these targets we have identified may indeed be clinically relevant. Hence, we demonstrated that a detailed ALF cellular decoding may enable pathway-specific therapeutic intervention.
In this work we used single cell RNA sequencing to identify heterogeneity of the innate lymphoid cell population in the liver and the effects of microbiota. We collected data from 3320 innate lymphoid cells (ILCs) form three conditions, germ free, antibiotics treated and conventional specific pathogen free mice. To perform single cell RNAseq of innate lymphoid cells we cleaned intestines, surgically removed adipose tissue and Peyer’s patches. We performed tissue digestion and cell isolation using Lamina Propria Dissociation kit from Miltenyi Biotech. Cell suspension was stained with antibodies against CD45, CD3, CD19, Gr-1, NKp46, CD127, and KLRG-1. Single cells were sorted into wells of 384 well plates and sequencing libraries were prepared using MARSseq protocol. The libraries were sequenced using Ilumina Nextseq. To analyse the data, we used the R package “MetaCell” this allowed to infer sub-clusters of cells within the canonical ILC subsets, their functional annotation and the interactions between cells.
Furthermore, I used the acetaminophen and thioacetamide ALF models to characterise over 40.000 single cell transcriptomes and to define the ALF cellular atlas. Using hierarchical clustering I identified 49 distinct populations and described their molecular states. Then I performed analysis of secretome and cell-to-cell interactions. The analysis of activation patterns revealed a common signature of 82-genes and further examination of promoters of these showed that MYC is a transcriptional regulator of this signature. MYC is driving expression of Ccl2 in stellate, Kupffer and endothelial cells which in turn drives infiltration of monocytes. Then we validated this using MYC inhibitors and flow cytometry, activity of liver enzymes in serum, histology and single cell sequencing. Then we performed experiments in germ free and antibiotics treated mice to examine the role of microbiota on the transcriptomic responses using single cell RNA sequencing and we were able to show that in absence of microbiota the responses are ameliorated and additionally there was less infiltration of monocytes. I validated this with flow cytometry, activity of liver enzymes in serum and histology. We hypothesised that microbiota as well as damage signal through the same pathway, i.e. TLR signalling and we used MyD88 Trif double knock out mice which do not have TLR signalling, because MyD88 and Trif are adaptor proteins of TLRs. With single cell RNA sequencing we found that in disease MyD88 Trif double knock out mice phenocopy MYC inhibition, suggesting that indeed TLR signalling is upstream MYC. We also tested inhibitors of MAPK pathway proteins (IRAK4, p38, TAK1, TPL2, RIP1 and ERK) in mouse disease models to find out which of the proteins mediate the signalling cascade and used flow cytometry to measure the extent of monocyte infiltration and liver damage with activity of liver enzymes in serum and histology.
Furthermore, I used the acetaminophen and thioacetamide ALF models to characterise over 40.000 single cell transcriptomes and to define the ALF cellular atlas. Using hierarchical clustering I identified 49 distinct populations and described their molecular states. Then I performed analysis of secretome and cell-to-cell interactions. The analysis of activation patterns revealed a common signature of 82-genes and further examination of promoters of these showed that MYC is a transcriptional regulator of this signature. MYC is driving expression of Ccl2 in stellate, Kupffer and endothelial cells which in turn drives infiltration of monocytes. Then we validated this using MYC inhibitors and flow cytometry, activity of liver enzymes in serum, histology and single cell sequencing. Then we performed experiments in germ free and antibiotics treated mice to examine the role of microbiota on the transcriptomic responses using single cell RNA sequencing and we were able to show that in absence of microbiota the responses are ameliorated and additionally there was less infiltration of monocytes. I validated this with flow cytometry, activity of liver enzymes in serum and histology. We hypothesised that microbiota as well as damage signal through the same pathway, i.e. TLR signalling and we used MyD88 Trif double knock out mice which do not have TLR signalling, because MyD88 and Trif are adaptor proteins of TLRs. With single cell RNA sequencing we found that in disease MyD88 Trif double knock out mice phenocopy MYC inhibition, suggesting that indeed TLR signalling is upstream MYC. We also tested inhibitors of MAPK pathway proteins (IRAK4, p38, TAK1, TPL2, RIP1 and ERK) in mouse disease models to find out which of the proteins mediate the signalling cascade and used flow cytometry to measure the extent of monocyte infiltration and liver damage with activity of liver enzymes in serum and histology.
We identified 6 different subtypes of innate lymphoid cells and through modelling cell-to-cell interactions between these subtypes, we identified known as well as putative novel modes of cell communication. Moreover, I observed shifts in population structure dependent on the microbiota, namely more ILC1 cells in conventional colonised mice than in antibiotics treated or germ free mice and more of ILC3p cell subset in germ free mice in comparison to colonised and antibiotics treated mice. The fact that enrichment of ILC3p is observable only in germ free condition, but not in antibiotics treated mice it suggests that this subset is developmentally determined by microbiota in early life. Differential gene expression analysis revealed terms related to immune system function as well as MAPK signalling. Moreover, we identified and characterised three cellular states in the liver - activated stellate cells, activated endothelial cells and activated Kupffer cells. I was able to demonstrate that these unique, previously uncharacterized stellate, endothelial, Kupffer cell, monocyte and neutrophil subsets drive a conserved chain of events leading to ALF.We unraveled a common Myc-dependent transcriptional program driving stellate, endothelial and Kupffer cell activation during ALF, which is regulated by microbiome through TLR and MAPK signalling. I characterised the populations and interactions between them, showing that Myc regulates transcription of Ccl2 chemokine that mediates signalling between resident cell types and blood monocytes to drive tissue infiltration of the latter population. We have also shown that abrogation of this transcriptional program by systemic administration of Myc, IRAK4 and p38 kinase inhibitors, or microbiome depletion, leads to significant ALF attenuation.