Monocytes and macrophages, with a focus of Crohn's disease
Abstract: Monocytes and macrophages are part of the body’s first line of defense, eliminating pathogens by phagocytosis or by releasing a broad array of inflammatory mediators, such as cytokines, chemokines and proteases. In humans, three subsets of monocytes are described with seemingly different functions. The classical (CD14++CD16-) monocytes, the intermediate (CD14++CD16+) monocytes and the non-classical (CD14+CD16++). In Crohn’s disease (CD) and other chronic diseases CD16+ monocytes are found to be increased in blood. Though the role of the three monocyte subsets in health and disease are not fully elucidated. Intestinal tissue resident and inflammatory macrophages are distinguished by low and high expression of CD14, respectively. The origin of human intestinal macrophages and their blood monocytic counterparts have been an unresolved paradigm. Thus, the overall aim of this thesis was to study the heterogeneity of monocyte subsets, their function and resemblance to their intestinal macrophage counterparts in CD and to investigate the potential of human cystatin C, a protease inhibitor, as a regulator of monocyte responses and a potential therapeutic candidate for CD. In this thesis, classical blood monocytes were found to be decreased in CD patients compared to healthy controls. Of the three subsets, classical monocytes were found to exert the most pro-inflammatory response by secreting the cytokines, IL-1β, IL-6, and TNF-α as well as MMP-1 upon pathogenic stimulation, mimicking microbial components found in CD. Especially immune complex activation elicited a pro-inflammatory response by high TNF-α and low IL-10 release. Classical monocytes also possessed highest capability to migrate in response to the chemokine CCL2 and showed highest phenotypic resemblance to an intestinal macrophage population, which we described as CD14hiHLA-DRdim. In all, we described three intestinal macrophage subsets, CD14hiHLA-DRdim, CD14hiHLA-DRbright and CD14loHLA-DRint, of which only the CD14hiHLA-DRdim was found to be increased in inflamed CD mucosa. However, further analysis of genetic variation among monocytes and the intestinal macrophages could be useful in discovering new target cells. Single cell gene-expression analysis was therefore tested on monocytes from a healthy donor. A hitherto undescribed multimodal gene expression and inter-cellular genetic variation of selected immune response genes were found. In order to dampen the immunologic response by monocytes/macrophages in CD we also investigated cystatin C as a potential immunomodulator and therapeutic target. We found that cystatin C decreased IL-1β and TNF-α released from pathogenic activated monocytes. Cystatin C decreased phosphorylation of the MAPK pathway ERK, and especially lowered levels of TNF-α in lamina propria cell cultures from CD patients. Together these results suggest that specific monocytes migrate to the intestine during inflammation in CD, and that cystatin C has the potential to decrease inflammatory mediators released from monocytes. Moreover, single cell gene-expression will allow further discovery of the heterogeneity within human monocytes and intestinal macrophages, potentially unravel new target cells.
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