Projects per year
Abstract
The intestine is a unique environment which can broadly be separated into the small intestine (SI) and colon. These segments are responsible for specific functions, such as nutrient absorption in the SI and waste disposal, water reabsorption and hosting the commensal microbiota in the colon. The intestine has immune effector sites such as the lamina propria (LP), where immune cells maintain tolerance and fight infection, and immune inductive sites where adaptive immune responses are initiated. Residing in the gut wall, the gut associated lymphoid tissues (GALT) consist of isolated lymphoid follicles (ILF) and Peyer’s patches, and are inductive sites for mucosal immunity.
Mononuclear phagocytes (MNP) are important immune cells that bridge the innate and adaptive immune system. They consist of two major subsets: macrophages and conventional dendritic cells (cDC). Macrophages are tissue resident cells and have important functions in clearing apoptotic cells, tissue remodelling and clearance of pathogens. They can arise from embryonic progenitors before birth, or from bone marrow derived monocytes. cDCs are derived from specific precursors in the bone marrow and patrol tissues to sample antigen; upon activation, they migrate to lymphoid tissue to prime naïve T cells.
The intestinal tissues also contain multiple cell types collectively termed mesenchymal stromal cells (MSC) which provide support for the epithelium lining the lumen and produce proteins for the LP’s structural matrix.
The aim of this thesis was to unravel the heterogeneity of the human intestinal MNP compartment. We approached this by isolating GALT and LP separately from healthy intestinal tissue taken from individuals undergoing colorectal cancer surgery, using a novel protocol developed recently in the Agace lab. After sorting for MNPs, the cells were subjected to single cell RNA sequencing (scRNA-seq) to unbiasedly characterize all intestinal MNPs. scRNA-seq was also applied to murine MSC LP from SI and colon to characterize MSC diversity and ontogeny.
In the first manuscript, I show that there are three distinct subtypes of cDCs in the LP of both human SI and colon. These are cDC1, cDC2 and the more recently discovered cDC3, which has similarities to cDC2. To examine the expression of surface proteins, we used CITE-seq antibodies and found that cDC2 could be enriched as CD1c+ CD207+ CD11a-, whereas cDC3 were CD1c+ CD207- CD11a+. These phenotypic markers allowed us to show that SI was relatively enriched in CD207+ CD11a- cDC2, while the colon had a higher frequency of CD207-CD11a+ cDC3. We also identified a putative common precursor for all the cDC in the LP, as well as downstream committed precursors for each cDC lineage which become increasingly similar to their mature counterparts.
Resident macrophages in the intestine are mainly derived from circulating monocytes. In the first manuscript, I was able to recreate this process by bioinformatic analysis and also showed that there are multiple distinct populations of mature macrophages. These may have different functions or they may have different locations within the LP. Furthermore, in inflammatory bowel disease the proportional distribution of monocytes and macrophages changed; the frequency of mature macrophages was reduced due to an influx of monocytes.
In the second manuscript, I compared the MNP populations in LP with GALT and observed that all major subsets of cDCs and macrophages were present in both locations. However, GALT, especially Peyer’s patches, had a higher frequency of cDCs than the surrounding LP, while the LP had a higher abundance of mature macrophages. By examining LP and GALT from a patient with Crohn’s disease, we found substantial recruitment of monocytes in inflamed tissues and this was associated with the appearance of a pro-inflammatory monocyte subset that was only rarely observed in healthy tissues.
In the third manuscript, I contributed to the bioinformatic analysis of MSC subsets in the murine intestine and together, the results from my thesis illustrate how unbiased computational approaches can be used to unravel cellular heterogeneity in the intestine. Our results add to the growing knowledge on cDCs, macrophage and stromal populations in healthy intestinal tissues, providing insights into their ontogeny, functions and behaviour during inflammation that may help develop new therapies.
Mononuclear phagocytes (MNP) are important immune cells that bridge the innate and adaptive immune system. They consist of two major subsets: macrophages and conventional dendritic cells (cDC). Macrophages are tissue resident cells and have important functions in clearing apoptotic cells, tissue remodelling and clearance of pathogens. They can arise from embryonic progenitors before birth, or from bone marrow derived monocytes. cDCs are derived from specific precursors in the bone marrow and patrol tissues to sample antigen; upon activation, they migrate to lymphoid tissue to prime naïve T cells.
The intestinal tissues also contain multiple cell types collectively termed mesenchymal stromal cells (MSC) which provide support for the epithelium lining the lumen and produce proteins for the LP’s structural matrix.
The aim of this thesis was to unravel the heterogeneity of the human intestinal MNP compartment. We approached this by isolating GALT and LP separately from healthy intestinal tissue taken from individuals undergoing colorectal cancer surgery, using a novel protocol developed recently in the Agace lab. After sorting for MNPs, the cells were subjected to single cell RNA sequencing (scRNA-seq) to unbiasedly characterize all intestinal MNPs. scRNA-seq was also applied to murine MSC LP from SI and colon to characterize MSC diversity and ontogeny.
In the first manuscript, I show that there are three distinct subtypes of cDCs in the LP of both human SI and colon. These are cDC1, cDC2 and the more recently discovered cDC3, which has similarities to cDC2. To examine the expression of surface proteins, we used CITE-seq antibodies and found that cDC2 could be enriched as CD1c+ CD207+ CD11a-, whereas cDC3 were CD1c+ CD207- CD11a+. These phenotypic markers allowed us to show that SI was relatively enriched in CD207+ CD11a- cDC2, while the colon had a higher frequency of CD207-CD11a+ cDC3. We also identified a putative common precursor for all the cDC in the LP, as well as downstream committed precursors for each cDC lineage which become increasingly similar to their mature counterparts.
Resident macrophages in the intestine are mainly derived from circulating monocytes. In the first manuscript, I was able to recreate this process by bioinformatic analysis and also showed that there are multiple distinct populations of mature macrophages. These may have different functions or they may have different locations within the LP. Furthermore, in inflammatory bowel disease the proportional distribution of monocytes and macrophages changed; the frequency of mature macrophages was reduced due to an influx of monocytes.
In the second manuscript, I compared the MNP populations in LP with GALT and observed that all major subsets of cDCs and macrophages were present in both locations. However, GALT, especially Peyer’s patches, had a higher frequency of cDCs than the surrounding LP, while the LP had a higher abundance of mature macrophages. By examining LP and GALT from a patient with Crohn’s disease, we found substantial recruitment of monocytes in inflamed tissues and this was associated with the appearance of a pro-inflammatory monocyte subset that was only rarely observed in healthy tissues.
In the third manuscript, I contributed to the bioinformatic analysis of MSC subsets in the murine intestine and together, the results from my thesis illustrate how unbiased computational approaches can be used to unravel cellular heterogeneity in the intestine. Our results add to the growing knowledge on cDCs, macrophage and stromal populations in healthy intestinal tissues, providing insights into their ontogeny, functions and behaviour during inflammation that may help develop new therapies.
Original language | English |
---|
Publisher | DTU Health Technology |
---|---|
Number of pages | 201 |
Publication status | Published - 2022 |
Fingerprint
Dive into the research topics of 'Heterogeneity in human intestinal mononuclear phagocytes'. Together they form a unique fingerprint.Projects
- 1 Finished
-
Heterogeneity in Human Intestinal Mononuclear Phagocytes
Christensen, L. W. (PhD Student), Haniffa, M. (Examiner), Agace, W. W. (Main Supervisor) & Olsen, L. R. (Supervisor)
01/04/2019 → 12/09/2022
Project: PhD