Thesis title: Novel mechanisms regulating CCRL2 expression and function
CCRL2 is a seven transmembrane domain receptor, structurally and functionally related to the atypical chemokine receptors family. However, the only recognized ligand for this receptor is the chemotactic protein chemerin. To date, two main functions were described to be associated to CCRL2. The first one is to act as presenting molecule when expressed on the surface of epithelial barrier cells, where it can concentrate chemerin, thus creating a membrane-bound chemerin gradient able to regulate the migration of adjacent leukocytes expressing the chemerin functional receptor Chemerin1. On the other hand, CCRL2 has been shown to regulate leukocytes migration through the interaction with conventional chemokine receptors. Indeed, by forming heterodimers, CCRL2 can regulate the functionality of the prototypical neutrophil chemokine receptor CXCR2. CCRL2 functionality was also investigated in-vivo in models of inflammatory diseases and carcinogenesis, providing important insights of its role in different pathological conditions.
Part I
Recently, a novel possible function for CCRL2 was described: the interaction with TLR4. This interaction is functional to induce the retention of TLR4 on the plasma membrane of tumor associated macrophages, thus promoting the maintenance of a pro-inflammatory TLR4-mediated signaling, preventing the switch towards a M2-like anti-inflammatory phenotype. Thus, TAMs could retain a pro-inflammatory anti-tumoral M1-like phenotype.
The first part of this thesis will elucidate the functional relationship between CCRL2 and other TLRs, specifically TLR7. The functional interaction between CCRL2 and TLR7 was investigated by using an in vitro model of bone-marrow derived dendritic cells (BMDCs) generated by CD34+ hematopoietic bone marrow precursors, obtained from WT and CCRL2 knockout (KO) mice. We focused our attention on dendritic cells (DCs) as they are the main antigen presenting cells, playing a crucial role in bridging innate and adaptive immune responses. WT and CCRL2 KO BMDCs were then stimulated with TLR7 agonist R848, with the purpose of performing a phenotypical and functional characterization. Our results displayed an impaired secretion of pro-inflammatory cytokines in CCRL2 KO DCs after TLR7 triggering, suggesting CCRL2 may be involved in the regulation of TLR7 mediated pro-inflammatory signaling. Moreover, on the one hand, flow cytometry experiments showed that the impaired pro-inflammatory secretion was not due to a defective maturation of CCRL2 KO cells. On the other hand, qPCR and Western Blot experiments demonstrated that CCRL2 does not affect TLR7 expression. Thus, given the crucial role of NF-κB in the production of pro-inflammatory cytokines, NF-κB translocation to the nucleus was assessed via Western Blot. The translocation of NF-κB to the nucleus was found impaired in CCRL2 KO BMDCs compared to WT BMDCs. These results suggest CCRL2 may play a crucial role in the NF-κB phosphorylation, thus impairing the pro-inflammatory response in DCs and affecting the secretion of pro-inflammatory cytokines in the supernatant.
Part II
Colorectal cancer (CRC) is the fourth most deadly and the third most common cancer worldwide with approximately 900’000 diagnosis per year, accounting around 10% of cancer-related deaths worldwide. Despite the role of CCRL2 in carcinogenesis is still under investigation, in a murine model of lung cancer it was demonstrated that its ablation reduced the recruitment of several myeloid cells subsets and NK cells in the tumor microenvironment, thus positioning CCRL2 as a tumor-suppressor gene. Inflammation represents one of the key drivers of CRC and preliminary data, generated in our laboratory, related to AOM/DSS murine model of inflammation-associated carcinogenesis, have shown that CCRL2 KO mice are more susceptible to the development of intestinal polyps in comparison to WT control animals. Moreover, previous results suggest a correlation between low CCRL2 expression and poorer survival rate in patients. Taken together, these findings suggest that CCRL2 may play a role as a novel tumor-suppressing gene, whose silencing promotes carcinogenesis.
One of the most relevant mechanisms of tumor-suppressor gene silencing is represented by DNA methylation. On this basis the aim of the second part of this thesis was to investigate CCRL2 methylation-mediated silencing in CRC. The model in which we decided to investigate CCRL2 methylation in CRC were human CRC cell lines that expressed (RKO) or did not express (SW480) CCRL2. The level of methylation of CCRL2 was investigated by two different approaches: an indirect method consisting in the treatment with 5-AZA-dC (a demethylating agent) and a direct method based on the immunoprecipitation of methylated DNA (MeDIP). Our results showed high levels of methylated DNA in the transcription factors binding site (TFBS) region of CCRL2 putative promoter in SW480 cells. In contrast, RKO cells, which were found to express CCRL2 in normal conditions, showed low levels of methylated DNA in the TFBS region. In fact, the treatment with 5-AZA-dC, led to an increase of CCRL2 expression in SW480 cells, but not in RKO cells.
Moreover, we set the basis for the analysis of histone modifications in CCRL2 TFBS region by chromatin immunoprecipitation (ChIP), targeting the tri-methylated fourth lysine residue of H3 histone (H3K4me3), a modification associated to transcriptional activation. In accordance with the results obtained by MeDIP, SW480 cells showed lower levels of H3K4me3 compared to RKO cells. Thus, our results support the hypothesis of CCRL2 epigenetic silencing in the context of CRC and lay the foundation to investigate the role of CCRL2 as a novel tumor-suppressor gene.