ROLE OF INTESTINAL CX3CR1+ MACROPHAGES IN THE DEVELOPMENT OF COLITIS-ASSOCIATED CANCER: FOCUS ON THE RELEVANCE OF THE MICROBIOME AND HEME-OXYGENASE-1
Creators: name: MARELLI, GIULIA
Publisher: name: Universit� degli Studi di Milano
Year: 2016

description: Gut represents a complex landscape in which commensal bacteria, harmless antigens and food proteins are strictly in contact with the immune system that has to be able to maintain a balance between the immune response and tolerance. In this context, mononuclear phagocytes are the most abundant population and macrophages represent a key player in this process of discrimination by allowing a peaceful coexistence. If this crucial checkpoint is lost, immune system is activated and falls in a dangerous and prolonged inflammation. Recent studies have described CX3CR1+ cells as a population of resident macrophages able to sample the gut lumen and to produce the anti-inflammatory cytokine IL-10 in order to maintain homeostasis. These macrophages are characterized by the expression of the chemokine receptor CX3CR1; however, how this receptor is involved in the molecular mechanisms that regulate the inflammatory response is largely unknown. The global aim of this thesis is to define the role of mucosal CX3CR1+ macrophages during acute and chronic inflammation and in the process of intestinal carcinogenesis, and to investigate the molecular mechanisms by which these immune cells maintain gut homeostasis. We analyzed the behavior of these macrophages in mice lacking the CX3CR1 receptor (CX3CR1GFP/GFP) and in wild type heterozygous mice (CX3CR1GFP/+). Firstly, we characterized them as a macrophagic population expressing CX3CR1, F4/80, CD11b and CD64 confirming the data present in literature. As the behavior of these cells is less defined during the resolution of inflammation, we performed a model of recovery from colitis and we found that KO mice displayed higher signs of inflammation compared to WT mice, and they were not able to recover as WT mice did. From the molecular point of view, we found that the inflammatory mediators were up-regulated in KO mice as well as members of the IL-10 family cytokines. The only mediator resulting down-regulated in KO mice was heme-oxygenase-1 (hemox-1). Hemox-1 is an anti-inflammatory enzyme over-expressed during tissue injury that is able to promote bacteria clearance. This regulation of this molecule is very complex and involves also IL-10. However, despite KO mice produced more IL-10, in an attempt to switch off inflammation, they were not able to produce an adequate amount of hemox-1. We reproduced, in vitro, the model of colitis and we showed that macrophages from KO mice responded to the inflammatory stimulus (LPS) with an aberrant response, up-regulating both pro and anti-inflammatory mediators, but not hemox-1. Moreover, we observed a synergism between LPS and the chemokine Fracktalkine/CX3CL1 (FKN) in the production of hemox-1 in WT mice and also found that stimulation with FKN alone was sufficient to produce hemox-1 in WT mice, unveiling a previously unidentified role of the chemokine receptor CX3CR1 in the regulation of hemox-1. Using the AOM/DSS model of colitis-induced carcinogenesis (CAC), a study that has never been performed in these genetically modified mice, we found significantly higher signs of inflammation in KO mice in terms of both cytokine production and immune cell infiltration, as well as higher score of tissue damage and number of polyps. Also in the CAC model, KO mice attempt to switch off inflammation by over-producing IL-10 levels; furthermore, hemox-1 was under-expressed. We therefore decided to enhance hemox-1 production using cobalt protoporphyrin IX (coPP), a compound able to stimulate hemox-1 in the intestine. The experiments revealed that treatment with coPP was able to revert the phenotype in KO mice, resulting in marked lower inflammation and reduced tumor load. To corroborate these findings, we investigated a genetic model of intestinal tumorigenesis (APCmin mice) and generated APCmin CX3CR1GFP/+ WT mice and APCmin CX3CR1GFP/GFP KO mice. Also in this tumor model, mice deficient for the CX3CR1 receptor demonstrated higher inflammation, lower Hemox-1 and more extensive tumorigenesis, thus overall confirming the results obtained with the sporadic CAC model. Hemox-1 acts on macrophages by stimulating the processes of phagocytosis; accordingly, we found that bacteria phagocytosis was significantly impaired in KO mice. The sequencing of the microbiota in the stools of mice treated with the AOM-DSS model, revealed a different microbiota composition, in particular, Akkermansia were down represented in KO mice. Akkermansia is reported to be impaired during inflammatory bowel diseases, leading to the rise of other bacteria and to an overall dysregulation of the total microbiome composition. This dysbiosis might be involved in disease relapse and rise of chronic inflammation that can lead to cancer development. Overall, we unveiled a new pathway by which intestinal CX3CR1+ macrophages operate to maintain gut homeostasis. If the CX3CL1-CX3CR1 checkpoint is lost, the FKN-induced production of hemox-1 is missing and an aberrant inflammatory response could persist and be corroborated by a state of dysbiosis. In this landscape, chronic inflammation and the impaired resolution could, over time, promote tumor development. Finally, we confirmed that treatment with the chemical compound coPP up-regulates hemox-1 also in the absence of functional CX3CR1 receptor, and drastically ameliorates the intestinal disease. These findings open a new scenario in the use of hemox-1-inducing drugs to restore the correct intestinal homeostasis in a therapeutic context.